Lanthionine synthetase c-like 2-based therapeutics

ABSTRACT

Provided are compounds that target the lanthionine synthetase C-like protein 2 pathway. The compounds can be used to treat a number of conditions, including infectious disease, autoimmune disease, diabetes, and a chronic inflammatory disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims priority under 35 USC § 119(e) to U.S.Provisional Patent Application 62/068,322 filed Oct. 24, 2014, and U.S.Provisional Patent Application 62/101,164 filed Jan. 8, 2015, theentirety of each of which is incorporated herein by reference.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made partially with U.S. Government support from theUnited States National Institutes of Health under SBIR grant1R43DK097940-01A1 and STTR grant 1R41DK099027-01A1 awarded toBioTherapeutics Inc. The U.S. Government has certain rights in theinvention.

FIELD OF THE INVENTION

The present invention relates to the field of medical treatments fordiseases and disorders. More specifically, the present invention relatesto classes of biologically active compounds that treat and preventinflammatory and immune mediated diseases such as inflammatory boweldisease, rheumatoid arthritis, psoriasis, multiple sclerosis, and type 1diabetes, as well as chronic inflammatory diseases and disorders such asinsulin resistance, impaired glucose tolerance, prediabetes, type 2diabetes, and obesity-related inflammation, among others.

BACKGROUND

Lanthionine C-like protein 2 (LANCL2) (also called “lanthioninesynthetase C-like protein 2” or “lanthionine synthetase component C-likeprotein 2”) is a signaling pathway protein that is expressed immunecells, gastrointestinal tract, neurons, testis and pancreas [1].Activating the LANCL2 pathway increases insulin sensitivity and reducesinflammation associated with various autoimmune, inflammatory andmetabolic conditions. Results of in vivo and in vitro testing in miceshowed that using compounds targeting this pathway reduce glucose levels2× in glucose tolerance tests as compared to controls and providedequivalent levels to prescription AVANDIA® (GlaxoSmithKline plc,Brentford, England)—an effective treatment but with significantside-affects. Targeting the LANCL2 pathway also reduces gut inflammationby 90% with a corresponding 4× reduction in the number of lesions. Theresults from this testing and other validations of the pathway arepublished in 12 peer-reviewed journal articles [2-13].

Within the category of autoimmune-related inflammation there iscurrently a global pandemic of autoimmune disorders such as inflammatorybowel disease (IBD), systemic lupus, rheumatoid arthritis, type 1diabetes, psoriasis, multiple sclerosis. There is also a pandemic ofchronic metabolic inflammatory diseases including metabolic syndrome,obesity, prediabetes, cardiovascular disease, and type 2 diabetes.Current treatments are moderately effective but are expensive and haveserious side effects. The route of administration for the most effectivetreatments for autoimmune diseases, such as anti-TNF antibodies, is viaIV or subcutaneous injection, requiring visits to clinics/surgeries andfrequent monitoring. The unique mode of action of LANCL2 provides fororally administered therapeutics that are as effective as anti-TNFantibodies but without the side effects and high cost. Given theepidemic of inflammatory and autoimmune diseases as a whole, the LANCL2pathway has the potential to significantly impact millions of patients.

Abscisic acid (“ABA”) is one of the natural compounds found in theoriginal screening process that binds to LANCL2.

There is an enormous number of compounds described in the field ofsynthetic organic chemistry. Various compounds are provided by thefollowing references: WO1997/036866 to Diana et al., WO 2006/053109 toSun et al., WO 2006/080821 to Kim et al., WO 2007/019417 to Nunes etal., WO 2009/067600 and WO 2009/067621 to Singh et al., WO 2008/079277to Adams et al., JP 2008/056615 to Urasoe et al., WO 2011/066898 toStoessel et al., US 2013/0142825 to Bassaganya-Riera et al., and U.S.Pat. No. 7,741,367 to Bassaganya-Riera et al. Some of the compoundsdescribed in these references are known to activate the LANCL2 pathwayand others are not.

There is a need to develop novel ligands of the LANCL2 pathway to allowtreatments to be tailored specifically to individual diseases and topotentially maximize their efficacy.

This application therefore describes a series of classes of compoundsthat have been developed by novel medicinal chemistry approaches, andscreened using in silico, in vitro, and in vivo techniques, to maximizetheir ability to bind to the LANCL2 protein and thus to effect abeneficial response in various disease conditions, including but notlimited to, autoimmune, chronic inflammatory, metabolic, and infectiousdiseases.

SUMMARY OF THE INVENTION

The invention provides compounds comprising formula Z—Y-Q-Y′—Z′ or apharmaceutically acceptable salt or ester thereof,

wherein:

-   -   Z is:

-   -   Y is:

-   -   Q is piperazine-1,4-diyl;        2,5-diazabicyclo[2.2.1]heptane-2,5-diyl;        2,5-diazabicyclo[2.2.2]octane-2,5-diyl; 1,4-diazepane-1,4-diyl;        benzene-1,4-diamine-N¹,N⁴-diyl; ethane-1,2-diamine-N¹,N²-diyl;        N¹,N²-dialkylethane-1,2-diamine-N¹,N²-diyl;        propane-1,3-diamine-N¹,N³-diyl;        N¹,N³-dialkylpropane-1,3-diamine-N¹,N³-diyl;        1,4-diaminoanthracene-9,10-dione-1,4-diyl; C₆        arene-1,4-diamine-N¹,N⁴-diyl wherein the arene is substituted        with one to four substituents in the 2, 3, 5, or 6 positions and        wherein the substituents are independently selected from the        group consisting of —C(O)O(C₁ to C₆)alkyl, OH, O(C₁ to C₆)alkyl,        (C₁ to C₆)alkyl, CF₃, F, Cl, and Br; or substituted        piperazine-1,4-diyl wherein the piperazine is substituted with        one to eight substituents in the 2, 3, 5, or 6 positions and        wherein the substituents are independently selected from the        group consisting of (C₁ to C₆)alkyl, aryl, aryl(C₁ to C₆)alkyl,        C(O)OH, and C(O)O(C₁ to C₆)alkyl;    -   Y¹ is:

or a single bond; and

-   -   Z′ is:

or R⁵;

wherein:

Y′ is a single bond only when Z′ is R5;

A₁ and A₁′ are each independently N, N(C₁ to C₆)alkyl, O, S, or CR⁶;

A₂ and A₂′ are each independently N or CR⁷;

A₃ and A₃′ are each independently NR⁸, O, or S;

A₄ and A₄′ are each independently N or CR⁹;

A₅ and A₅′ are each independently N or CR¹⁰;

A₆ and A₆′ are each independently N or CR¹¹;

R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰,and R¹¹ are each independently selected from the group consisting ofhydrogen; alkyl; halo; trifluoromethyl; dialkylamino wherein each alkylis independently selected; —NH₂; alkylamino; arylalkyl; heteroarylalkyl;heterocycloalkyl; substituted heterocycloalkyl substituted with 1 to 2substituents independently selected from the group consisting of—C(O)OH, —C(O)O(C₁ to C₆)alkyl, (C₁ to C₆)alkyl, —CF₃, F, Cl, and Br;and substituted heteroarylalkyl;

-   -   wherein the substituted heteroarylalkyl is substituted with 1 to        3 substituents independently selected from the group consisting        of —NH₂; —NH(C₁ to C₆)alkyl; —N((C₁ to C₆)alkyl)₂ wherein each        alkyl is independently selected; alkyl; halo; aryl; substituted        aryl substituted with 1 to 3 substituents independently selected        from the group consisting of —SO₂R¹², —OR¹³, -halo, —CN, —CF₃,        aminoalkyl-, —S(O)R¹⁴, and alkyl; heterocycloalkyl; heteroaryl;        substituted aryl substituted with 1 to 3 substituents        independently selected from the group consisting of alkyl, —CF₃,        F, Cl, and Br; alkylamino-; heterocycloalkyl-alkyl-amino-;        alkylaminoalkylamino-; —NHC(O)OR¹⁵; —NHC(O)NR¹⁶R¹⁷;        —C(O)NR¹⁶R¹⁷; and substituted heteroaryl substituted with 1 to 3        substituents selected from the group consisting of alkyl, halo,        CN, NH₂, —NH(C₁-C₆ alkyl), —N(C₁-C₆ alkyl)₂ wherein each alkyl        is independently selected, —CF₃, and substituted aryl        substituted with 1 to 3 substituents independently selected from        the group consisting of —S(O)₂R¹⁵ and —CN;        -   wherein R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, and R¹⁷ are each            independently selected from the group consisting of C₁-C₆            alkyl, dialkylamino comprising independently selected C₁-C₆            alkyl, —NH₂, alkylamino, heterocycloalkyl, and substituted            heterocycloalkyl substituted with one to two substituents            independently selected from the group consisting of            —C(O)O(C₁-C₆ alkyl) and C₁-C₆ alkyl.            In some compounds, at least one of A₃ and A₃′ is O or S. In            some compounds, one or both of A₁ and A₁′ is N. In some            compounds, one or both of A₂ and A₂′ is CH, A₃ is NH, A₄ is            N, A₅ is CH, and A₆ is CH. In some compounds, one or both of            A₂ and A₂′ is CH, one or both of A₃ and A₃′ is NH, one or            both of A₄ and A₄′ is N, one or both of A₅ and A₅′ is CH,            and one or both of A₆ and A₆′ is CH. In some compounds, Q is            piperazine-1,4-diyl;            2,5-diazabicyclo[2.2.1]heptane-2,5-diyl;            2,5-diazabicyclo[2.2.2]octane-2,5-diyl;            1,4-diazepane-1,4-diyl;            N¹,N²-dialkylethane-1,2-diamine-N¹,N²-diyl;            N¹,N³-dialkylpropane-1,3-diamine-N¹,N³-diyl;            1,4-diaminoanthracene-9,10-dione-1,4-diyl; C₆            arene-1,4-diamine-N¹,N⁴-diyl wherein the arene is            substituted with one to four substituents in the 2, 3, 5, or            6 positions and each substituent is independently selected            from the group consisting of —C(O)O(C₁ to C₆)alkyl, OH, O(C₁            to C₆)alkyl, (C₁ to C₆)alkyl, CF₃, F, Cl, and Br; or            substituted piperazine-1,4-diyl wherein the piperazine is            substituted with one to eight substituents in the 2, 3, 5,            or 6 positions and each substituents is independently            selected from the group consisting of (C₁ to C₆)alkyl, aryl,            aryl(C₁ to C₆)alkyl, C(O)OH, and C(O)O(C₁ to C₆)alkyl.

In some compounds, the formula Z—Y-Q-Y′—Z′ is:

or salts thereof. In some compounds, members of one or more pairsselected from the group consisting of A₁ and A₁′, A₂ and A₂′, A₃ andA₃′, A₄ and A₄′, A₅ and A₅′, A₆ and A₆′, R₁ and R₁′, R₂ and R₂′, R₃ andR₃′, and R₄ and R₄′ are the same. In some compounds, members of one ormore pairs selected from the group consisting of A₁ and A₁′, A₂ and A₂′,A₃ and A₃′, A₄ and A₄′, A₅ and A₅′, A₆ and A₆′, R₁ and R₁′, R₂ and R₂′,R₃ and R₃′, and R₄ and R₄′ are different. In some compounds, members ofeach pair selected from the group consisting of A₁ and A₁′, A₂ and A₂′,A₃ and A₃′, A₄ and A₄′, A₅ and A₅′, A₆ and A₆′, R₁ and R₁′, R₂ and R₂′,R₃ and R₃′, and R₄ and R₄′ are the same. In some compounds, members ofeach pair selected from the group consisting of A₁ and A₁′, A₂ and A₂′,A₃ and A₃′, A₄ and A₄′, A₅ and A₅′, A₆ and A₆′, R₁ and R₁′, R₂ and R₂′,R₃ and R₃′, and R₄ and R₄′ are different.

In some compounds, the formula Z—Y-Q-Y′—Z′ is:

orsalts thereof.

Some compounds of the invention have the structure of:

or

salts thereof.

The invention also provides compounds comprising formula A-B-C or apharmaceutically acceptable salt or ester thereof,

wherein:

-   -   A is:

-   -   B is:

and

-   -   C is:

wherein:

-   -   A₇, A₈, A₉, A₁₀, A₁₁, A₁₂, A₁₃, and A₁₄ are each independently        selected from CH, CR¹⁸, and N;    -   A₁₅, A₁₆, A₁₇, A₁₈, A₁₉, and A₂₀ are each independently selected        from CH, CR¹⁹, N, NR²⁰, O, and S, with the proviso that only one        of A₁₅, A₁₆, and A₁₇ can be N, NR²⁰, O, or S and only one of        A₁₈, A₁₉, and A₂₀ can be N, NR²⁰, O, or S;    -   R¹⁸ and R¹⁹ are each independently selected from C₁-C₆ alkyl;        C₁-C₆ dialkylamino, wherein each C₁-C₆ alkyl is independently        selected; —NH₂; alkylamino; heterocycloalkyl; and substituted        heterocycloalkyl, wherein the substituted heterocycloalkyl is        substituted with one to two substituents independently selected        from the group consisting of: —C(O)O(C₁-C₆ alkyl) and C₁-C₆        alkyl; wherein in compounds with more than one CR¹⁸ each R¹⁸ is        independently selected, and in compounds with more than one CR¹⁹        each R¹⁹ is independently selected; and    -   R²⁰ is C₁-C₆ alkyl.        In some compounds B is:

Some compounds have a structure of:

or salts thereof.

The invention also provides methods of treating a condition in an animalwith any one or more of the compounds described herein. The methodscomprise administering an effective amount of one or more of thecompounds described herein to the animal. The condition may be selectedfrom the group consisting of an infectious disease, an autoimmunedisease, diabetes, and a chronic inflammatory disease. In some methods,the infectious disease comprises a viral disease, such as influenzainfection. In some methods, the autoimmune disease comprises anautoimmune inflammatory disease, such as inflammatory bowel disease,including ulcerative colitis and/or Crohn's disease. In some methods,the diabetes is selected from the group consisting of type 1 diabetesand type 2 diabetes. In some methods, the chronic inflammatory diseasecomprises metabolic syndrome. In some methods, the methods compriseadministering an amount of a compound effective to increase activity ofLANCL2, decrease inflammation, and/or increase anti-inflammatoryeffects.

The invention also provides compounds for use in treating a condition inan animal with any one or more of the compounds described herein. Thecompounds for such use include any compounds described herein. The usemay comprise administering an effective amount of one or more of thecompounds described herein to the animal, wherein the condition isselected from the group consisting of an infectious disease, anautoimmune disease, diabetes, and a chronic inflammatory disease. Insome versions, the infectious disease comprises a viral disease, such asinfluenza infection. In some versions, the autoimmune disease comprisesan autoimmune inflammatory disease, such as inflammatory bowel disease,including ulcerative colitis and/or Crohn's disease. In some versions,the diabetes is selected from the group consisting of type 1 diabetesand type 2 diabetes. In some versions, the chronic inflammatory diseasecomprises metabolic syndrome. In some versions, the compound iseffective to increase activity of LANCL2, decrease inflammation, and/orincrease anti-inflammatory effects.

The objects and advantages of the invention will appear more fully fromthe following detailed description of the preferred embodiment of theinvention made in conjunction with the accompanying drawings.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A and 1B. Computational prediction of binding of compounds toLANCL2 and biochemical experimental validation using SPR.

FIG. 2. Clustering histogram for the top five clusters of NSC6160. Onehundred docking runs were performed with NSC6160 docked to LANCL2 usingAutoDock Tools. The RMSD cluster tolerance was 2 Å. Binding energies arelisted in kJ/mol.

FIG. 3. Clustering histogram for the top five clusters of ABA. Onehundred docking runs were performed with ABA docked to LANCL2 usingAutoDock Tools. The RMSD cluster tolerance was 2 Å. Binding energies arelisted in kJ/mol.

FIG. 4. Clustering histogram for the top five clusters of BT-11. Onehundred docking runs were performed with BT-11 docked to LANCL2 usingAutoDock Tools. The RMSD cluster tolerance was 2 Å. Binding energies arelisted in kJ/mol.

FIG. 5. Clustering histogram for the top five clusters of BT-6. Onehundred docking runs were performed with BT-6 docked to LANCL2 usingAutoDock Tools. The RMSD cluster tolerance was 2 Å. Binding energies arelisted in kJ/mol.

FIG. 6. Clustering histogram for the top five clusters of BT-15. Onehundred docking runs were performed with BT-15 docked to LANCL2 usingAutoDock Tools. The RMSD cluster tolerance was 2 Å. Binding energies arelisted in kJ/mol.

FIG. 7. Clustering histogram for the top five clusters of BT-ABA-5a. Onehundred docking runs were performed with BT-ABA-5a docked to LANCL2using AutoDock Tools. The RMSD cluster tolerance was 2 Å. Bindingenergies are listed in kJ/mol.

FIG. 8. Binding kinetics of lanthionine synthetase C-like protein 2(LANCL2) with BT-11 and BT-15. Panels A and C show surface plasmonresonance (SPR) sensorgrams for the binding of varying concentrations ofBT-11 (A) and BT-15 (C) to immobilized LANCL2. Panels B and D show plotsof maximal resonance unit (RU) versus concentration of BT-11 (B) andBT-15 (D). Steady state dissociation constants (K_(D)) utilizing a 1:1binding model are indicated.

FIGS. 9A and 9B. Binding kinetics of lanthionine synthetase C-likeprotein 2 (LANCL2) with BT-6 (FIG. 9A) and BT-ABA-5a (FIG. 9B). Surfaceplasmon resonance (SPR) sensorgrams for the binding of varyingconcentrations of BT-6 and BT-ABA-5a to immobilized LANCL2 are shown.

FIG. 10. Effect of oral administration on disease activity and grosspathology of mice with dextran sodium sulfate (DSS) colitis. Panel Ashows disease activity index scores in mice treated with either BT-11 orvehicle only. Panels B-C show gross pathology scores from the (B)spleen, (C) mesenteric lymph nodes (MLN), and (D) colon in mice treatedwith either vehicle or BT-11. Statistically significant differences(P<0.05) are indicated with an asterisk (n=10).

FIG. 11. Effect of oral BT-11 administration on colonic inflammatorylesions in mice with DSS colitis. Representative micrographs of (A,D)control (B, E) DSS, and (C, F) BT-11 treated DSS mice are shown.Histopathological lesions were evaluated based on (G) leukocyticinfiltration, (H) epithelial erosion, and (I) mucosal thickening.Statistically significant differences (P<0.05) are indicated with anasterisk (n=10).

FIG. 12. Dose-Response effect of oral BT-11 administration on colonicinflammatory lesions in mice with DSS colitis. Histopathological lesionswere evaluated based on (A) leukocytic infiltration, (B) mucosalthickening, and (C) epithelial erosion. Statistically significantdifferences (P<0.05) are indicated with an asterisk (n=10).

FIG. 13. Colonic gene expression analysis of TNFα, interleukin 10(IL-10) and LANCL2. Colonic gene expression to assess the levels of (A)proinflammatory TNFα, (B) IL-10, and (C) LANCL2 are shown. Statisticallysignificant differences (P<0.05) are indicated with an asterisk (n=10).

FIG. 14. Dose-Response effect of oral administration of BT-11 on colonicpro- and anti-inflammatory immune cell subsets in mice with DSS colitis.Flow cytometry analyses were used to measure (A) TNFα+ cells, (B)IL-10+CD4+ T cells, and (C) FOXP3+CD4+ T cells in the colonic mucosa.

FIG. 15. Effect of oral BT-11 administration on tissue gross pathologylesions in wild-type and LANCL2−/− mice with DSS colitis. Panel A showsdisease activity index scores in wild-type versus LANCL2−/− mice treatedwith either BT-11 or vehicle only. Panels B-D show gross pathologyscores from the (B) colon, (C) mesenteric lymph nodes (MLN), and (D)spleen in wild-type and LANCL2−/− mice treated with either vehicle orBT-11. Statistically significant differences (P<0.05) are indicated withan asterisk (n=10).

FIG. 16. Effect of oral BT-11 administration on colonic inflammatorylesions in wild-type and LANCL2−/− mice with DSS colitis.Histopathological lesions were evaluated based on (A) leukocyticinfiltration, (B) mucosal thickening and (C) epithelial erosion.Statistically significant differences between groups (P<0.05) areindicated with an asterisk.

FIG. 17. Effect of oral BT-11 administration on immune cell subsetsinfiltrating the colonic lamina propria, spleen and mesenteric lymphnodes (MLN) of wild-type and LANCL2−/− mice with chronic colitis. Flowcytometry was used to assay the levels of (A) colonic MCP1+CD45+ cells,(B) MCP1+CD45+ cells in the MLN, (C) colonic TNFα+CD45+ cells, (D)colonic MHC-II+CD11c+ granulocytes, (E) colonic IL-10+CD45+ cells, and(F) IL-10+CD45+ splenocytes after treatment with BT-11. Statisticallysignificant differences between groups (P<0.05) are indicated with anasterisk.

FIG. 18. Effect of oral BT-11 administration on disease activity index(DAI) scores in IL-10−/− mice with chronic colitis. DAI scores on IL-10null mice that developed spontaneous colitis and that were treated dailywith either vehicle alone or with 20, 40, and 80 mg of BT-11/Kg bodyweight (n=10). Statistically significant differences between groups(P<0.05) are indicated with an asterisk.

FIG. 19. Effect of oral BT-11 administration on macroscopic tissuescoring in a chronic model of colitis after treatment with BT-11.Macroscopic scores in (A) spleen, (B) mesenteric lymph nodes (MLN), and(C) colon of mice treated with either vehicle or BT-11 at threedifferent concentrations (20, 40, and 80 mg/Kg). Statisticallysignificant differences between groups (P<0.05) are indicated with anasterisk.

FIG. 20. Effect of oral BT-11 administration on colonichistopathological lesions in chronic IL-10−/− model of IBD.Histopathological lesions were evaluated based on (A) leukocyticinfiltration, (B) epithelial erosion, and (C) mucosal thickening.Statistically significant differences between groups (P<0.05) areindicated with an asterisk.

FIG. 21. Effect of oral BT-11 administration on immune cell subsetsinfiltrating the colonic lamina propria of IL-10−/− with chroniccolitis. Flow cytometry was used to assay the levels of (A) F4/80+macrophages, (B) MHC-II+CD11c+ dendritic cells (DC), (C) CD4+ FOXP3+regulatory T cells, and (D) T helper 1 (Th1) cells in the colonic LPafter treatment with BT-11. Statistically significant differencesbetween groups (P<0.05) are indicated with an asterisk.

FIG. 22. Effect of oral BT-11 administration on immune cell subsetsinfiltrating the spleen and mesenteric lymph nodes of IL-10−/− withchronic colitis. Flow cytometry was used to assay the levels of (A) CD4+RORgt+ T cells, (B) CD4+ FOXP3+ T cells, (C) CD4+CD45+ FOXP3+ regulatoryT cells, and (D) T helper 1 (Th1) cells after treatment with BT-11.Statistically significant differences between groups (P<0.05) areindicated with an asterisk.

FIG. 23. Effect of oral treatment with BT-11 on colonic expression ofLANCL2 and TNFα. Colonic gene expression was used to assess the levelsof (A) LANCL2 and (B) TNFα. Statistically significant differencesbetween groups (P<0.05) are indicated with an asterisk.

FIG. 24. Effect of oral BT-11 administration on disease activity indexscores in vehicle versus treated mice in an adoptive transfer model ofchronic colitis. RAG2−/− mice were treated with vehicle or BT-11following transfer of 400,000 naïve CD4+ T cells intraperitoneally.Statistically significant differences between groups (P<0.05) areindicated with an asterisk.

FIG. 25. Effect of oral BT-11 administration on disease activity indexscores in vehicle versus treated wild-type versus LANCL2−/− transferredmice in an adoptive transfer model of chronic colitis. RAG2−/− mice weretreated with vehicle or BT-11 following transfer of 400,000 naïve CD4+ Tcells intraperitoneally from either wild-type or LANCL2−/− donors.Statistically significant differences between groups (P<0.05) areindicated with an asterisk.

FIG. 26. Effect of oral BT-11 administration on weight loss in thechronic IBD model of CD4+-induced colitis. Mice were weighed andpercentage of weight loss was calculated. Statistically significantdifferences between groups (P<0.05) are indicated with an asterisk.

FIG. 27. Effect of oral BT-11 administration on macroscopic tissuescoring in a chronic model of CD4+ T cell-induced colitis aftertreatment with BT-11. Macroscopic scores in (A) spleen, (B) MLN, (C)colon, and (D) ileum of mice treated with either vehicle or BT-11 at 80mg/Kg are shown. Statistically significant differences between groups(P<0.05) are indicated with an asterisk.

FIG. 28. Effect of oral BT-11 administration on macroscopic tissuescoring in a chronic model of CD4+ T cell-induced colitis with wild-typeand LANCL2−/− mice after treatment with BT-11. Macroscopic scores in (A)colon, (B) MLN, and (C) spleen of wild-type and LANCL2−/− mice treatedwith either vehicle or BT-11 at 80 mg/Kg are shown. Statisticallysignificant differences between groups (P<0.05) are indicated with anasterisk.

FIG. 29. Effect of oral BT-11 administration on colonic and ilealhistopathology in vehicle versus treated mice in an adoptive transfermodel of chronic colitis. Histopathological lesions in the colon (A, C,E) and ileum (B, D, F) were evaluated based on (A, B) leukocyticinfiltration, (C, D) epithelial erosion, and (E, F) mucosal thickening.Statistically significant differences between groups (P<0.05) areindicated with an asterisk.

FIG. 30. Effect of oral BT-11 administration on colonic histopathologyin vehicle versus treated mice transferred with either wild-type orLANCL2−/−CD4+ T cells in an adoptive transfer model of chronic colitis.Histopathological lesions were evaluated based on (A) leukocyticinfiltration, (B) mucosal thickening, and (C) epithelial erosion.Statistically significant differences between groups (P<0.05) areindicated with an asterisk.

FIG. 31. Effect of oral BT-11 administration on disease activity indexscores in vehicle versus treated mice in an adoptive transfer model ofchronic colitis. Flow cytometry was used to assay the levels of (A)F4/80+CD11b+ macrophages, (B) CD45+ IFNg+ cells, (C) CD4+ FOXP3+regulatory T cells, and (D) CD4+IL-10+ anti-inflammatory cells aftertreatment with BT-11. Statistically significant differences betweengroups (P<0.05) are indicated with an asterisk.

FIG. 32. Effect of oral BT-11 administration on disease activity indexscores in vehicle versus treated mice in an adoptive transfer model ofchronic colitis. Flow cytometry was used to assay the levels of (A) CD4+FOXP3+ T cells, (B) CD4+IL-10+ T cells, (C) CD45+ IFNg+ cells in theMLN, and (D) CD4+ FOXP3+ T cells, (E) CD4+IL-10+ T cells, (F) CD45+IFNg+ cells in the spleen after treatment with BT-11. Statisticallysignificant differences between groups (P<0.05) are indicated with anasterisk.

FIG. 33. Effect of oral BT-11 administration on disease activity indexscores in vehicle versus treated wild-type versus PPARγ−/− transferredmice in an adoptive transfer model of chronic colitis. RAG2−/− mice weretreated with vehicle or BT-11 following transfer of 400,000 naïve CD4+ Tcells intraperitoneally from either wild-type or PPARγ−/− donors. (A)Disease activity index scores versus time post-transfer are shown.Histopathological lesions in the colon were evaluated based on (B)leukocytic infiltration, (C) mucosal thickening, and (D) epithelialerosion. Statistically significant differences between groups (P<0.05)are indicated with an asterisk.

FIG. 34. Effect of oral BT-11 administration on fasting blood glucoseand insulin levels in NOD mice with diabetes. (A) Fasting glucose levelswere assessed at weeks 0, 1, 3, 4, 5, 10, and 11 of treatment withvehicle or BT-11 (80 mg/kg/d). (B) Fasting serum insulin levels wereassessed at week 5 of treatment with either vehicle or BT-11 (80mg/kg/d). Statistically significant differences (P<0.05) are indicatedwith an asterisk (n=10).

FIG. 35. Effect of oral BT-11 administration in lesion formation in thepancreas of type 1 diabetic mice. Histopathological lesions wereevaluated based on leukocytic infiltration, lesion formation, and tissueerosion. Statistically significant differences between groups (P<0.05)are indicated with an asterisk.

FIG. 36. Effect of oral BT-11 administration on (A) fasting bloodglucose levels and (B) glucose tolerance test. (A) Mice were fasted for12 h and blood glucose levels were assessed at weeks 2 and 12 afterexperiment set up. (B) Mice were also challenged with an IP glucoseinjection (2 g/Kg) and glucose was measured. Statistically significantdifferences (P<0.05) are indicated with an asterisk.

FIG. 37. Effect of oral BT-11 administration on pro-inflammatorypopulations infiltrating into the white adipose tissue (WAT). WAT wasexcised and digested and immunophenotyping results were assessed by flowcytometry. Levels of (A) infiltrating macrophages and (B) Ly6c^(high)GR1+ infiltrating cells are shown. Statistically significant differences(P<0.05) are indicated with an asterisk.

FIG. 38. Effect of oral BT-11 administration on glucose homeostasis in adb/db model of diabetes. (A) Fasting blood glucose (FBG) concentrationsfrom leptin receptor-deficient (db/db) mice treated with either BT-11 orvehicle at weeks 1 and 3 after experiment set up are shown. (B) Plasmaglucose levels after intraperitoneal glucose challenge (1 g/Kg bodyweight) are shown. Blood was collected before (0), then 15, 30, 60, 90,120, 180, 220, and 265 minutes after glucose load. Statisticallysignificant differences between groups (P<0.05) are indicated with anasterisk.

FIG. 39. Effect of oral BT-11 administration on expression of LANCL2,TNFα, and MCP-1 in white adipose tissue (WAT) from mice withdiet-induced obesity. Gene expression analysis of LANCL2, TNFα, andMCP-1 was evaluated compared to the untreated mice. The line at zerorepresents the baseline of mice that received vehicle only.

FIG. 40. Effect of oral BT-11 administration on clinical scores andmorbidity of mice infected with Influenza virus. Mice were infected withinfluenza virus and clinically scored throughout the experiment.Clinical scores were noted for (A) activity and (B) physical appearance.(C) The percentage of mice that lost more than 15% of body weight wasplotted to show changes in morbidity. Statistically significantdifferences between groups (P<0.05) are indicated with an asterisk.

DETAILED DESCRIPTION OF THE INVENTION General Definitions

Unless otherwise stated, the following definitions are used throughoutthe present application:

Analysis of Variance (ANOVA): Arithmetic process for partitioning theoverall variation in data sets into specific components based on sourcesof variation. It has been used to determine whether numericaldifferences between treatment groups are statistically significant.

Adipogenesis: The process by which new adipocytes or fat storage cellsare generated.

Allele: One of a number of viable DNA coding of the same gene.

Conjugated diene: A molecule containing two double bonds separated by asingle bond.

Db/db mice: Term used to define a type of mouse which lacks both allelesof a long isoform of leptin receptor. This deficiency results in a highpredisposition to developing type 2 diabetes. See examples below forfurther discussions on Db/db mice.

Enantiomer: Optical isomer; chemical classification of molecules basedon their ability to rotate the plain of polarization clockwise (+) oranti-clockwise (−).

Glycemia: Concentration of glucose in blood.

Hyperglycemia: Increased concentrations of glucose in blood beyond thenormal ranges.

Hyperinsulinemia: Increased concentrations of insulin in blood beyondthe normal ranges.

Insulinemia: Concentration of insulin in blood.

Insulin resistance: Inability of tissues to respond to insulin and takeup glucose from the blood.

Substantially pure: Having a purity of at least 90% by weight,preferably at least 95% by weight such as at least 98%, 99% or about100% by weight.

Type 2 diabetes or non-insulin dependent diabetes mellitus: Termreferring to a common type of diabetes caused by an unresponsiveness ofcells to the actions of insulin. If the cells do not respond to insulin,they are unable to take up glucose from blood, which results inglucotoxicity. In addition, the cells are deprived from the energyderived from glucose oxidation.

IBD: Inflammatory bowel disease (IBD) involves chronic inflammation ofall or part of your digestive tract. IBD primarily includes ulcerativecolitis and Crohn's disease. Both usually involve severe diarrhea, pain,fatigue and weight loss. IBD can be debilitating and sometimes leads tolife-threatening complications.

Ulcerative colitis (UC): UC is an IBD that causes long-lastinginflammation and sores (ulcers) in the innermost lining of your largeintestine (colon) and rectum.

Crohn's Disease: Crohn's disease is an IBD that cause inflammation ofthe lining of your digestive tract. In Crohn's disease, inflammationoften spreads deep into affected tissues. The inflammation can involvedifferent areas of the digestive tract—the large intestine, smallintestine or both.

IL-10: Interleukin-10 (IL-10), also known as human cytokine synthesisinhibitory factor (CSIF), is an anti-inflammatory cytokine. In humans,IL-10 is encoded by the IL10 gene.

FOXP3: FOXP3 (forkhead box P3) also known as scurfin is a proteininvolved in immune system responses. A member of the FOX protein family,FOXP3 appears to function as a master regulator (transcription factor)in the development and function of regulatory T cells.

TNF-alpha: Tumor necrosis factor (TNF, cachexin, or cachectin, andformerly known as tumor necrosis factor alpha or TNFα) is cytokineinvolved in systemic inflammation and is a member of a group ofcytokines that stimulate the acute phase reaction.

MCP1: Monocyte chemoattractant protein-1. An older term for a CCcytokine which is critical for development of atherosclerotic lesions,found in endothelial cells, macrophages and in vascular smooth musclecells of patients undergoing coronary artery bypass procedures. Theofficially preferred term is now chemokine (C-C motif) ligand 2.

Interferon gamma: Interferon gamma is a pro-inflammatory dimerizedsoluble cytokine that is the only member of the type II class ofinterferons.

Type 1 diabetes: Type 1 diabetes, once known as juvenile diabetes orinsulin-dependent diabetes, is a chronic condition in which the pancreasproduces little or no insulin, a hormone needed to allow sugar (glucose)to enter cells to produce energy.

Leukocytic infiltration: Leukocyte infiltration refers to the process ofmoving or infiltrating of the leukocytes into the injured tissue tobegin the repair process.

Chemical Definitions

The term “alkyl,” by itself or as part of another substituent, means,unless otherwise stated, a fully saturated, straight, branched chain, orcyclic hydrocarbon radical, or combination thereof, and can include di-and multi-valent radicals, having the number of carbon atoms designated(e.g., C₁-C₁₀ means from one to ten carbon atoms, inclusive). Examplesof alkyl groups include, without limitation, methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)ethyl, cyclopropylmethyl, and homologs, and isomers thereof,for example, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Theterm “alkyl,” unless otherwise noted, also includes those derivatives ofalkyl defined in more detail below as “heteroalkyl” and “cycloalkyl.”

The term “alkenyl” means an alkyl group as defined above except that itcontains one or more double bonds. Examples of alkenyl groups includevinyl, 2-propenyl, crotyl, 2-isopentenyl, 2-(butadienyl),2,4-pentadienyl, 3-(1,4-pentadienyl), etc., and higher homologs andisomers.

The term “alkynyl” means an alkyl or alkenyl group as defined aboveexcept that it contains one or more triple bonds. Examples of alkynylgroups include ethynyl, 1- and 3-propynyl, 3-butynyl, and the like,including higher homologs and isomers.

The terms “alkylene,” “alkenylene,” and “alkynylene,” alone or as partof another substituent means a divalent radical derived from an alkyl,alkenyl, or alkynyl group, respectively, as exemplified by—CH₂CH₂CH₂CH₂—.

Typically, alkyl, alkenyl, alkynyl, alkylene, alkenylene, and alkynylenegroups will have from 1 to 24 carbon atoms. Those groups having 10 orfewer carbon atoms are preferred in the present invention. The term“lower” when applied to any of these groups, as in “lower alkyl” or“lower alkylene,” designates a group having 10 or fewer carbon atoms.

“Substituted” refers to a chemical group as described herein thatfurther includes one or more substituents, such as lower alkyl, aryl,acyl, halogen (e.g., alkylhalo such as CF₃), hydroxy, amino, alkoxy,alkylamino, acylamino, thioamido, acyloxy, aryloxy, aryloxyalkyl,mercapto, thia, aza, oxo, both saturated and unsaturated cyclichydrocarbons, heterocycles and the like. These groups may be attached toany carbon or substituent of the alkyl, alkenyl, alkynyl, alkylene,alkenylene, and alkynylene moieties. Additionally, these groups may bependent from, or integral to, the carbon chain itself.

The term “aryl” is used herein to refer to an aromatic substituent,which may be a single aromatic ring or multiple aromatic rings which arefused together, linked covalently, or linked to a common group such as adiazo, methylene or ethylene moiety. The common linking group may alsobe a carbonyl as in benzophenone. The aromatic ring(s) may include, forexample phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenone,among others. The term “aryl” encompasses “arylalkyl” and “substitutedaryl.” For phenyl groups, the aryl ring may be mono-, di-, tri-, tetra-,or penta-substituted. Larger rings may be unsubstituted or bear one ormore substituents.

“Substituted aryl” refers to aryl as just described including one ormore functional groups such as lower alkyl, acyl, halogen, alkylhalo(e.g., CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy,phenoxy, mercapto, and both saturated and unsaturated cyclichydrocarbons which are fused to the aromatic ring(s), linked covalentlyor linked to a common group such as a diazo, methylene, or ethylenemoiety. The linking group may also be a carbonyl such as in cyclohexylphenyl ketone. The term “substituted aryl” encompasses “substitutedarylalkyl.”

The term “halogen” or “halo” is used herein to refer to fluorine,bromine, chlorine, and iodine atoms.

The term “hydroxy” is used herein to refer to the group —OH.

The term “amino” is used to designate NRR′, wherein R and R′ areindependently H, alkyl, alkenyl, alkynyl, aryl, or substituted analogsthereof. “Amino” encompasses “alkylamino,” denoting secondary andtertiary amines, and “acylamino” describing the group RC(O)NR′.

Administration

In the course of the methods of the present invention, a therapeuticallyeffective amount of compounds of the invention can be administered to ananimal, including mammals and humans, in many ways. While in thepreferred embodiment, the compounds of the invention are administeredorally or parenterally, other forms of administration such as throughmedical compounds or aerosols are also contemplated.

For oral administration, the effective amount of compounds may beadministered in, for example, a solid, semi-solid, liquid, or gas state.Specific examples include tablet, capsule, powder, granule, solution,suspension, syrup, and elixir agents. However, the compounds are notlimited to these forms.

To formulate the compounds of the invention into tablets, capsules,powders, granules, solutions, or suspensions, the compound is preferablymixed with a binder, a disintegrating agent and/or a lubricant. Ifnecessary, the resultant composition may be mixed with a diluent, abuffer, an infiltrating agent, a preservative and/or a flavor, usingknown methods. Examples of the binder include crystalline cellulose,cellulose derivatives, cornstarch, cyclodextrins, and gelatin. Examplesof the disintegrating agent include cornstarch, potato starch, andsodium carboxymethylcellulose. Examples of the lubricant include talcand magnesium stearate. Further, additives, which have beenconventionally used, such as lactose and mannitol, may also be used.

For parenteral administration, the compounds of the present inventionmay be administered rectally or by injection. For rectal administration,a suppository may be used. The suppository may be prepared by mixing thecompounds of the present invention with a pharmaceutically suitableexcipient that melts at body temperature but remains solid at roomtemperature. Examples include but are not limited to cacao butter,carbon wax, and polyethylene glycol. The resulting composition may bemolded into any desired form using methods known to the field.

For administration by injection, the compounds of the present inventionmay be injected hypodermically, intracutaneously, intravenously, orintramuscularly. Medicinal drugs for such injection may be prepared bydissolving, suspending or emulsifying the compounds of the inventioninto an aqueous or non-aqueous solvent such as vegetable oil, glycerideof synthetic resin acid, ester of higher fatty acid, or propylene glycolby a known method. If desired, additives such as a solubilizing agent,an osmoregulating agent, an emulsifier, a stabilizer, or a preservative,which has been conventionally used may also be added. While notrequired, it is preferred that the composition be sterile or sterilized.

To formulate the compounds of the invention into suspensions, syrups, orelixirs, a pharmaceutically suitable solvent may be used. Included amongthese is the non-limiting example of water.

The compounds of the invention may also be used together with anadditional compound having other pharmaceutically suitable activity toprepare a medicinal drug. A drug, either containing a compound of theinvention as a stand-alone compound or as part of a composition, may beused in the treatment of subjects in need thereof.

The compounds of the invention may also be administered in the form ofan aerosol or inhalant prepared by charging the compounds in the form ofa liquid or fine powder, together with a gaseous or liquid sprayingagent and, if necessary, a known auxiliary agent such as an inflatingagent, into a non-pressurized container such as an aerosol container ora nebulizer. A pressurized gas of, for example, dichlorofluoromethane,propane or nitrogen may be used as the spraying agent.

The compounds of the invention may be administered to an animal,including mammals and humans, in need thereof as a pharmaceuticalcomposition, such as tablets, capsules, solutions, or emulsions.Administration of other forms of the compounds described in thisinvention, including but not limited to esters thereof,pharmaceutically-suitable salts thereof, metabolites thereof,structurally related compounds thereof, analogs thereof, andcombinations thereof, in a single dose or a multiple dose, are alsocontemplated by the present invention.

The compounds of the invention may also be administered to an animal inneed thereof as a nutritional additive, either as a food ornutraceutical supplement.

The terms “preventing,” “treating,” or “ameliorating” and similar termsused herein, include prophylaxis and full or partial treatment. Theterms may also include reducing symptoms, ameliorating symptoms,reducing the severity of symptoms, reducing the incidence of thedisease, or any other change in the condition of the patient, whichimproves the therapeutic outcome.

The compounds described in this invention are preferably used and/oradministered in the form of a composition. Suitable compositions are,preferably, a pharmaceutical composition, a foodstuff, or a foodsupplement. These compositions provide a convenient form in which todeliver the compounds. Compositions of the invention may comprise anantioxidant in an amount effective to increase the stability of thecompounds with respect to oxidation or solubility.

The amount of compound that is administered in the method of theinvention or that is for administration in the use of the invention isany suitable amount. It is preferably from about 0.0001 g to about 20 g(more preferably 0.01 g to 1 g, such as 0.05 g to 0.5 g) of compound perday. Suitable compositions can be formulated accordingly. Those of skillin the art of dosing of biologically active agents will be able todevelop particular dosing regimens for various subjects based on knownand well understood parameters.

A preferred composition according to the invention is a pharmaceuticalcomposition, such as in the form of tablets, pills, capsules, caplets,multiparticulates (including granules, beads, pellets andmicro-encapsulated particles), powders, elixirs, syrups, suspensions,and solutions. Pharmaceutical compositions will typically comprise apharmaceutically acceptable diluent or carrier. Pharmaceuticalcompositions are preferably adapted for administration parenterally ororally. Orally administrable compositions may be in solid or liquid formand may take the form of tablets, powders, suspensions, and syrups,among other things. Optionally, the compositions comprise one or moreflavoring and/or coloring agents. In general, therapeutic andnutritional compositions may comprise any substance that does notsignificantly interfere with the action of the compounds on the subject.

Pharmaceutically acceptable carriers suitable for use in suchcompositions are well known in the art of pharmacy. The compositions ofthe invention may contain 0.01-99% by weight of the compounds of theinvention. The compositions of the invention are generally prepared inunit dosage form. Preferably the unit dosage of compounds described inthe present invention is from 1 mg to 1000 mg (more preferably from 50mg to 500 mg). The excipients used in the preparation of thesecompositions are the excipients known in the art.

Further examples of product forms for the composition are foodsupplements, such as in the form of a soft gel or a hard capsulecomprising an encapsulating material selected from the group consistingof gelatin, starch, modified starch, starch derivatives such as glucose,sucrose, lactose, and fructose. The encapsulating material mayoptionally contain cross-linking or polymerizing agents, stabilizers,antioxidants, light absorbing agents for protecting light-sensitivefills, preservatives, and the like. Preferably, the unit dosage ofcompounds in the food supplements is from 1 mg to 1000 mg (morepreferably from 50 mg to 500 mg).

In general, the term carrier may be used throughout this application torepresent a composition with which the compounds described may be mixed,be it a pharmaceutical carrier, foodstuff, nutritional supplement, ordietary aid. The materials described above may be considered carriersfor the purposes of the invention. In certain embodiments of theinvention, the carrier has little to no biological activity on thecompounds of the invention.

Dose: The methods of the present invention can comprise administering atherapeutically effective amount of compound to an animal in needthereof. The effective amount of compound depends on the form of thecompound administered, the duration of the administration, the route ofadministration (e.g., oral or parenteral), the age of the animal, andthe condition of the animal, including mammals and humans.

For instance, an amount of a compound effective to treat or prevent type2 diabetes, prediabetes, type 1 diabetes, impaired glucose tolerance,insulin resistance, ulcerative colitis, or Crohn's disease, or any othercondition described herein in an animal can range from 0.1-10,000mg/kg/day. A preferred effective amount of compound is 1 to 5,000mg/kg/day, with a more preferred dose being 2 to 100 mg/kg/day. Theupper limit of the effective amount to be administered is not critical,as the compounds are relatively non-toxic as our toxicology datademonstrates. The effective amount of compound is most effective intreating or preventing ulcerative colitis, Crohn's disease, type 2diabetes, type 1 diabetes, pre-diabetes, metabolic syndrome, impairedglucose tolerance, and insulin resistance of an animal when administeredto an animal for periods ranging from about 7 to 100 days, with apreferred period of 15 to 50 days, and a most preferred period of 30 to42 days.

An amount of compound most effective in preventing over-activation ofthe immune system can range from 0.1 to 500 mg/kg/day, with a preferreddose of 1 to 150 mg/kg/day.

When the effective amount of the compound of the present invention isadministered in a nutritional, therapeutic, medical, or veterinarycomposition, the preferred dose ranges from about 0.01 to 2.0% wt/wt tothe food or nutraceutical product.

In certain other embodiments, the present invention provides for use ofLANCL2-binding compounds and also structurally related compounds, suchas a compound selected from the group consisting the compound, estersthereof, pharmaceutically suitable salts thereof, metabolites thereof,structurally related compounds thereof, or combinations thereof in thetreatment and prevention of IBD and GI tract inflammation.

In addition, in general, the present invention relates to inhibition ofinflammation in the GI tract, wherein the relevant components includethe stomach, small intestine, large intestine, and rectum. The effectresults from the exposure of compound to various cells types in the bodythat induces a biological effect. The cells may include those from GItract tissues, immune cells (i.e. macrophages, monocytes, lymphocytes),or epithelial cells. In certain embodiments, the invention provides fortreating subjects with a compound of the invention, for example as adietary supplement, to reduce or prevent inflammation related toinflammatory bowel disease, either Crohn's Disease or UlcerativeColitis. The present invention also contemplates administering thecompounds of the invention to the GI tract in order to suppress theexpression of cellular adhesion molecules in the gut.

When practiced, the methods of the invention can be by way ofadministering the compounds to a subject via any acceptableadministration route using any acceptable form, as is described above,and allowing the body of the subject to distribute the compounds to thetarget cell through natural processes. As is described above,administering can likewise be by direct injection to a site (e.g.,organ, tissue) containing a target cell (i.e., a cell to be treated).

Furthermore, administering can follow any number of regimens. It thuscan comprise a single dose or dosing of experimental compound, ormultiple doses or dosings over a period of time. Accordingly, treatmentcan comprise repeating the administering step one or more times until adesired result is achieved. In certain embodiments, treating cancontinue for extended periods of time, such as weeks, months, or years.Those of skill in the art are fully capable of easily developingsuitable dosing regimens for individuals based on known parameters inthe art. The dosage amounts for compounds of the invention may be usedin the methods of these embodiments of the invention. For the treatmentof IBD, GI tract inflammation or suppressing expression of cellularadhesion molecules in the gut, it is preferred that the compounds beadministered at amounts of about 1 mg/day to 9,000 mg/day.

The amount to be administered will vary depending on the subject, stageof disease or disorder, age of the subject, general health of thesubject, and various other parameters known and routinely taken intoconsideration by those of skill in the medical arts. As a generalmatter, a sufficient amount of compound will be administered in order tomake a detectable change in the amount of inflammation in the GI tract,which with IBD is often related to the amount of pain an individual isexperiencing. With patients not currently experiencing IBD symptoms, thechange one might look for may involve immune cell parameters such asTNFα expression on immune-cells or the percent of regulatory T-cells inthe blood. Suitable amounts are disclosed herein, and additionalsuitable amounts can be identified by those of skill in the art withoutundue or excessive experimentation, based on the amounts disclosedherein.

In one aspect, the invention provides a method of treating or preventinga subject suffering from IBD, or otherwise healthy individuals, perhapswith a genetic predisposition for Crohn's Disease or ulcerative colitis,from developing IBD. The method may also involve treating those with aremissive form of IBD. According to the invention, the term “a subjectsuffering from IBD” is used to mean a subject (e.g., animal, human)having a disease or disorder showing one or more clinical signs that aretypical of IBD. In general, the method of treating or preventingaccording to this aspect of the invention comprises administering to thesubject an amount of compound therapy that is effective in treating orpreventing one or more symptoms or clinical manifestations of IBD, or inpreventing development of such symptom(s) or manifestation(s).

Thus, according to the methods of the invention, the invention canprovide methods of treatment of IBD, inflammation associated withenteric infection and inflammation associated with autoimmune diseases.The methods of treatment can be prophylactic methods. In certainembodiments, the method is a method of treating IBD, inflammationassociated with enteric infection and inflammation associated withautoimmune diseases. In other embodiments, the method is a method ofpreventing IBD. In embodiments, the method is a method of preventing aremissive form of IBD from becoming active. In still other embodiments,the method is a method of improving the health status of a subjectsuffering from IBD, inflammation associated with enteric infection andinflammation associated with autoimmune diseases. Organisms causinggastroenteric infections include but are not limited to: Escherichiacoli, Shigella, Salmonella, pathogenic Vibrios, Campylobacter jejuni,Yersina enterocolitica, Toxoplasma gondii, Entamoeba histolytica andGiardia lamblia. Accordingly, in certain embodiments, the inventionprovides a method of protecting the health, organs, and/or tissues of asubject suffering from IBD, inflammation associated with entericinfection and inflammation associated with autoimmune diseases or atrisk from developing IBD, inflammation associated with enteric infectionand inflammation associated with autoimmune diseases.

In one embodiment of the invention, the method of treating IBD comprisestreating IBD without causing discernable side-effects, such assignificant weight gain, systemic immune suppression, cushingoidappearance, osteopenia/osteoporosis, or pancreatitis that is common ofcurrently available IBD treatments (i.e. corticosteroids, tumor necrosisfactor alpha inhibitors). That is, it has been found that the method oftreating according to the present invention, which provides thetreatment effect, at least in part, by affecting the expression and/oractivation of LANCL2 in some cells, provides the beneficial effectwithout causing a significant gain in weight, for example by fluidretention, in the subject being treated, as compared to other similarsubjects not receiving the treatment.

As such, the methods of the present invention can provide methods ofreducing inflammation. The methods can reduce inflammation systemically(i.e., throughout the subject's body) or locally (e.g., at the site ofadministration or the site of inflammatory cells, including but notlimited to T cells and macrophages). In treating or preventinginflammation according to the methods of the present invention, oneeffect that may be seen is the decrease in the number of blood monocytesor macrophages and lymphocytes infiltrating the intestine. Another maybe the increase in regulatory immune cell populations, such as CD4⁺CD25⁺FoxP3⁺ regulatory T-cells, or an increase in regulatory properties oflymphocytes or macrophages (e.g. increased interleukin 4 (IL-4) or IL-10or decreased TNF-α and IL-6). Another may be the decreased presence ofinflammatory genes and/or adhesion molecules. The methods can thus alsobe considered methods of affecting or altering the immune response of asubject to whom the compound therapy is administered. The subject mayhave inflammatory bowel disease or another condition in which theimmunomodulation of T cells or downregulation of cellular adhesionmolecules is a desired outcome.

The invention also provides methods of treating an infectious diseasewith the compounds described herein. Non-limiting examples of suchinfectious diseases include viral infections, bacterial infections, andfungal infections.

Non-limiting examples of viral infections include infections fromviruses in the family adenoviridae, such as adenovirus; viruses in thefamily herpesviridae such as herpes simplex, type 1, herpes simplex,type 2, varicella-zoster virus, epstein-barr virus, humancytomegalovirus, human herpesvirus, and type 8; viruses in the familypapillomaviridae such as human papillomavirus; viruses in the familypolyomaviridae such as BK virus and JC virus; viruses in the familypoxviridae such as smallpox; viruses in the familyhepadnaviridae such ashepatitis B virus; viruses in the family parvoviridae such as humanbocavirus and parvovirus B19; viruses in the family astroviridae such ashuman astrovirus; viruses in the family caliciviridae such as norwalkvirus; viruses in the family picornaviridae such as coxsackievirus,hepatitis A virus, poliovirus, and rhinovirus; viruses in the familycoronaviridae such as acute respiratory syndrome virus; viruses in thefamily flaviviridae such as hepatitis C virus, yellow fever virus,dengue virus, and West Nile virus, viruses in the family togaviridaesuch as rubella virus; viruses in the family hepeviridae such ashepatitis E virus; viruses in the family retroviridae such as humanimmunodeficiency virus (HIV); viruses in the family orthomyxoviridaesuch as influenza virus; viruses in the family arenaviridae such asguanarito virus, junin virus, lassa virus, machupo virus, and sabiavirus; viruses in the family bunyaviridae such as Crimean-Congohemorrhagic fever virus; viruses in the family filoviridae such as ebolavirus and marburg virus; viruses in the family paramyxoviridae such asmeasles virus, mumps virus, parainfluenza virus, respiratory syncytialvirus, human metapneumovirus, hendra virus, and nipah virus; viruses inthe family rhabdoviridae such as rabies virus; unassigned viruses suchas hepatitis D virus; and viruses in the family reoviridae such asrotavirus, orbivirus, coltivirus, and banna virus, among others.

Non-limiting examples of bacterial infections include infections withthe bacteria described above, in addition to Bacillus anthracis,Bacillus cereus, Bordetella pertussis, Borrelia burgdorferi, Brucellaabortus, Brucella canis, Brucella melitensis, Brucella suisCampylobacter jejuni Chlamydia pneumoniae, Chlamydia trachomatis,Chlamydophila psittaci, Clostridium botulinum, Clostridium difficile,Clostridium perfringens, Clostridium tetani, Corynebacteriumdiphtheriae, Enterococcus faecalis, Enterococcus faecium, Escherichiacoli, Francisella tularensis, Haemophilus influenzae, Helicobacterpylori, Legionella pneumophila, Leptospira interrogans, Listeriamonocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis,Mycobacterium ulcerans, Mycoplasma pneumoniae, Neisseria gonorrhoeae,Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii,Salmonella typhi, Salmonella typhimurium, Shigella sonnei,Staphylococcus aureus, Staphylococcus epidermidis, Staphylococcussaprophyticus, Streptococcus agalactiae, Streptococcus pneumoniae,Streptococcus pyogenes, Treponema pallidum, Vibrio cholerae, Yersiniapestis, Yersinia enterocolitica, Yersinia pseudotuberculosis, and otherspecies from the genera of the above-mentioned organisms.

Non-limiting examples of fungal infections include infection with fungiof the genus Aspergillus, such as Aspergillus fumigatus, which causeaspergillosis; fungi of the genus Blastomyces, such as Blastomycesdermatitidis, which cause blastomycosis; fungi of the genus Candida,such as Candida albicans, which cause candidiasis; fungi of the genusCoccidioides, which cause coccidioidomycosis (valley fever); fungi ofthe genus Cryptococcus, such as Cryptococcus neoformans and Cryptococcusgattii, which cause cryptococcosis; dermatophytes fungi, which causeringworm; fungi that cause fungal keratitis, such as Fusarium species,Aspergillus species, and Candida species; fungi of the genusHistoplasma, such as Histoplasma capsulatum, which cause histoplasmosis;fungi of the order Mucorales, which cause mucormycosis; fungi of thegenus Saccharomyces, such as Saccharomyces cerevisiae; fungi of thegenus Pneumocystis, such as Pneumocystis jirovecii, which causepneumocystis pneumonia; and fungi of the genus Sporothrix, such asSporothrix schenckii, which cause sporotrichosis.

The invention also provides methods of treating an autoimmuneinflammatory disease with the compounds described herein. Non-limitingexamples of autoimmune inflammatory diseases include inflammatory boweldisease (IBD), systemic lupus, rheumatoid arthritis, type 1 diabetes,psoriasis, and multiple sclerosis, among others.

The invention also provides methods of treating chronic inflammatorydiseases with the compounds described herein. Non-limiting examples ofchronic inflammatory diseases includes metabolic syndrome, obesity,prediabetes, cardiovascular disease, and type 2 diabetes, among others.

The invention also provides methods of treating diabetes with thecompounds described herein, including type 1 diabetes, type 2 diabetes,and other types of diabetes. The term “diabetes” or “diabetes mellitus”is used to encompass metabolic disorders in which a subject has highblood sugar (i.e., hyperglycemia). Hyperglycemic conditions have variousetiologies, such as the pancreas does not produce enough insulin, orcells do not respond to the insulin that is produced. There are severalrecognized sub-types of diabetes. Type 1 diabetes is characterized bythe complete failure of the body to produce insulin or the failure ofthe body to produce enough insulin. Type 2 diabetes generally resultsfrom insulin resistance, a condition in which cells fail to use insulinproperly. Type 2 diabetes sometimes co-presents with an insulindeficiency. Gestational diabetes occurs when pregnant women without aprevious diagnosis of diabetes develop hyperglycemia. Less common formsof diabetes include congenital diabetes (due to genetic defects relatingto insulin secretion), cystic fibrosis-related diabetes, steroiddiabetes induced by high doses of glucocorticoids, and several forms ofmonogenic diabetes (including maturity onset diabetes of the young).Monogenic diabetes encompasses several hereditary forms of diabetescaused by mutations in a single, autosomal dominant gene (as contrastedto more complex, polygenic etiologies resulting in hyperglycemia).

In view of the above methods, it should be evident that the presentinvention provides LANCL2-binding compound therapy for use in contactingcells, such as in treating cells of a subject. The above discussionfocuses on the use of the compounds of the present invention as part ofa composition for use in what could generally be considered apharmaceutical or medical setting.

The compounds described in this invention for the treatment of IBD, GItract inflammation, and other conditions described may be formulated asa pharmaceutical, nutritional composition, functional food composition,or dietary aid, as are described in greater detail above.

The elements and method steps described herein can be used in anycombination whether explicitly described or not.

All combinations of method steps as used herein can be performed in anyorder, unless otherwise specified or clearly implied to the contrary bythe context in which the referenced combination is made.

As used herein, the singular forms “a,” “an,” and “the” include pluralreferents unless the content clearly dictates otherwise.

Numerical ranges as used herein are intended to include every number andsubset of numbers contained within that range, whether specificallydisclosed or not. Further, these numerical ranges should be construed asproviding support for a claim directed to any number or subset ofnumbers in that range. For example, a disclosure of from 1 to 10 shouldbe construed as supporting a range of from 2 to 8, from 3 to 7, from 5to 6, from 1 to 9, from 3.6 to 4.6, from 3.5 to 9.9, and so forth.

All patents, patent publications, and peer-reviewed publications (i.e.,“references”) cited herein are expressly incorporated by reference tothe same extent as if each individual reference were specifically andindividually indicated as being incorporated by reference. In case ofconflict between the present disclosure and the incorporated references,the present disclosure controls.

It is understood that the invention is not confined to the particularconstruction and arrangement of parts herein illustrated and described,but embraces such modified forms thereof as come within the scope of theclaims.

MOLECULAR MODELING EXAMPLES

Example 1: Molecular Modeling of LANCL2 Ligand Binding Introduction

Established LANCL2 μgonists such as abscisic acid (ABA) and NSC61610exert anti-inflammatory activity in a broad range of diseases modelsranging from IBD to diabetes and influenza. The value of LANCL2 as anovel therapeutic target merits efforts to discover and develop a newclass of orally active drugs for the treatment of chronic metabolic,immune-mediated, and infectious disease. As discussed in the presentexample, additional LANCL2 agonists were developed through rational drugdesign that iteratively combines computational modeling and experimentalvalidation. The present example shows approaches to increase rationaldrug design and medicinal chemistry efforts to increase solubility,increase binding to LANCL2, lower cost, and understand the LANCL2protein itself.

Methods Structure of LANCL2. No crystal structure for LANCL2 exists.Therefore in order to understand the structure and function of LANCL2,homology modeling of human LANCL2 was performed using the crystalstructure of LANCL1 as a template. Model quality was assessed andrefinements were made through energy minimization procedures. Homologymodeling predicts the 3D structure of a protein via identifying itshomologous proteins from other members of the protein family whosestructures have been solved experimentally [52]. When proteins have morethan 35% sequence identity, they are likely to be homologous. LANCL1shares 54% sequence identify with LANCL2 [15].

Compound Generation and Ligand Structure.

Structures of LANCL2 μgonists were generated (FIGS. 1A and 1B). SMILESof these agonists were generated using the NIH's online SMILESTranslator and Converter [53]. Concurrently, individual structural .pdbfiles were generated and downloaded. AutoDock Tools was using to convertpdb files into the .pdbqt necessary for virtual screening.

Virtual Screening.

The docking of the generated derivative files was performed withAutoDock Tools. A search space was defined, including grid box centerand x, y, and z dimensions. The docking applied to the whole proteintarget, with a grid covering the whole protein surface. The grid was aregular cuboid (77.8 Å×77.8 Å×77.8 Å) with grid points separated by0.608 Å. This grid was centered in the middle of the protein. Thesedimensions and spacing allowed the grid to cover the entire surface ofLANCL2. The genetic algorithm was used in stochastic globaloptimization. One hundred bound conformations were generated by AutoDockTools for each compound. The 100 resulting poses of each derivative wereclustered with an RMSD cluster tolerance of 2.0 Å.

Analyzing Virtual Screening Results.

The search for the best way to fit each compound into LANCL2 usingAutoDock Vina resulted in docking log files that contained records ofdocking, including binding energy of each predicted binding mode for allthe compounds. Binding energies represent the sum of the totalintermolecular energy, total internal energy and torsional free energyminus the energy of the unbound system. Compounds were ranked by themost negative energy value. The lowest binding energy pose in the firstcluster was considered as the most favorable docking pose. A lowerbinding free energy indicates a more stable protein-ligand system and ahigher affinity between protein and ligand. Exemplary compounds arefurther validated by in vitro testing and pre-clinical studies usingmouse models of human diseases.

Results

NSC61610 Docking Summary.

A histogram of NSC61610's top five clusters with the energy of thelowest energy position is given in FIG. 2. NSC61610 has very highaffinity for the ‘central cleft.’ The top two clusters, representing 7%of total runs, each direct to this site. Due to the two angstromtolerance, it is likely other clusters direct to this site. The next twoclusters direct to an ‘allosteric site’ near the blue random coil.

ABA Docking Summary.

A histogram of ABA's top five clusters with the energy of the lowestenergy position is given in FIG. 3. ABA has moderate affinity but veryhigh specificity for the ‘allosteric’ site between the light green helixand light green random coil. 29% of runs directed to this top cluster.The second cluster also directed to this site. Due to the two angstromtolerance, it is likely other cluster direct to this site. The fourthcluster appears to be in the ‘central cleft.’ This leaves open thequestion of the true therapeutic site of ABA.

BT-11 Docking Summary.

A histogram of BT-11's top five clusters with the energy of the lowestenergy position is given in FIG. 4. BT-11's top two clusters direct tothe ‘central cleft’ but represent only 2% of runs. However, due to thetwo angstrom tolerance, it is likely other clusters direct to this site.BT-11 has slightly less affinity for this site than NSC61610 but morethan ABA. BT-11 has demonstrated therapeutic efficacy (see examplesbelow).

BT-6 Docking Summary.

A histogram of BT-6's top five clusters with the energy of the lowestenergy position is given in FIG. 5. BT-6 has the highest affinity of anycompound docked. The top two, perhaps three, clusters direct to the‘central cleft.’ Due to the two angstrom tolerance, it is likely otherclusters direct to this site. Cluster 4 directs to the ‘allosteric’ sitealong the blue random coil.

BT-15 Docking Summary.

A histogram of BT-15's top five clusters with the energy of the lowestenergy position is given in FIG. 6. BT-15 does not have the bindingaffinity of either NSC61610 or BT-11. While it does appear to directtoward the ‘central cleft,’ this effect does not appear to be aspronounced as NSC61610 or BT-11.

BT-ABA-5a docking summary. A histogram of BT-ABA-5a's top five clusterswith the energy of the lowest energy position is given in FIG. 7.BT-ABA-5a's highest affinity is in a spot not seen in any previousdocking examined. However, clusters 2 and 3 represent the vast majorityof runs, at 32%. Cluster 2 directs to an allosteric site in the backright. Cluster 3 directs to the ‘allosteric’ site of ABA. Cluster 4 alsodirects to this site. Due to the two angstrom tolerance, it is likelyother clusters direct to this site.

Discussion

Both ABA and NSC61610 exert LANCL2-dependent immune-modulatory,anti-inflammatory, and anti-diabetic effects, however computationalpredictions suggest that they bind at different sites of LANCL2. Asexpected, the rationally designed ligands direct primarily to theprimary binding sites of ABA and NSC61610. The BT-ABA compounds aresmaller in size and have —COOH functional groups; it makes intuitivesense they would direct toward a hydrophilic surface pocket. The BTcompounds are much more hydrophobic; therefore it makes intuitive sensethey would direct to the more hydrophobic central cleft surrounded byalpha-helices.

The binding affinities have a moderate correlation with SPR data (FIGS.1A and 1B; see examples below). SPR data (with K_(D) value) suggests anorder of binding strength of NSC61610 (2.3 & 6.3), BT-11 (6.3 & 7.7),BT-15 (11.4 & 21.4), BT-6 (18.2). Modeling data (with lowest BE)suggests an order of binding strength of BT-6 (−10.47), NSC61610(−10.27), BT-11 (−9.39), BT-15 (−8.87). Besides the flip in BT-6 fromworst to first, SPR data and modeling data suggest the same order ofbinding strength. Molecular modeling data combined with rational drugdesign is likely to yield better understanding of the LANCL2 proteinwhich will allow for further development of analogs that target andactivate the LANCL2 pathway to exploit its potent anti-diabetic andanti-inflammatory properties.

MEDICINAL CHEMISTRY EXAMPLES Example 2: BT-11 and Salt

As shown in Scheme 2-1, A solution of6-(1H-Benzimidazol-2-yl)pyridine-2-carboxylic acid (12 g) in DMF (100mL) was cooled to 0° C., and then sequentially added EDC.HCl (1.5 eq),HOBt (1.5 eq) and DIPEA (1.2 eq, taken in volumes with densitypresumed). The mixture was stirred for 10 min at 0° C. Piperazine (0.5eq) was added and the reaction mixture was allowed to warm to RTgradually and stirred for 16 h. After completion of the reaction(monitored by TLC, eluent: 10% MeOH in DCM), the reaction mixture waspoured into ice-cold water (˜300 mL), the precipitated solid wasfiltered, washed with ice-cold water and dried to get BT-11 (10 g, 75%)as pale brown solid. ¹H NMR (400 MHz, DMSO-d6), δ 13.0 (s, 1H), 12.8 (s,1H), 8.38 (dd, 2H), 8.13 (dt, 2H), 7.73 (dd, 2H), 7.67 (d, 2H), 7.57(dd, 2H), 7.25 (m, 4H), 3.90 (bs, 2H), 3.80 (bdd, 2H), 3.65 (bdd, 2H),3.56 (bs, 2H). LCMS-ES 529.44 [M+H]⁺, 265.46 [(M+2H)/2]⁺⁺.

As shown in Scheme 2-2, a suspension of BT-11 (1.0 eq) in minimal amountof MeOH (5 mL) was cooled to 0° C., was added 4M methanolic HCl (excess,15 mL/1 g) dropwise over a period of 15-20 min. The mixture was allowedgradually to warm to RT for 3 h. After completion of the reaction(monitored by TLC, eluent: 10% MeOH in CH₂Cl₂), the volatiles wereevaporated under reduced pressure. The crude material was washed with10% MeOH in CH₂Cl₂ and lyophilized to get an off-white solid (850 mg,75%). ¹H NMR (400 MHz, DMSO-d₆), δ 8.58 (dd, 2H), 8.29 (dt, 2H), 7.83(m, 6H), 7.44 (bd, 4H), 3.91 (bs, 2H), 3.81 (bm, 2H), 3.64 (bm, 2H) 3.55(bs, 2H). LCMS-ES 529.56 [M+H]⁺.

Example 3: BT-12

As shown in Scheme 3-1, a solution of6-(benzoxazol-2-yl)pyridine-2-carboxylic acid (4.05 g) in 10% DMF inCH₂Cl₂ was treated with EDC.HCl (1.5 eq), HOBt (1.5 eq) and DIPEA (1.2eq, taken in volumes with density presumed) and 0.5 eq. of piperazine at0° C. The mixture was allowed to warm to RT for 16 h. A light brownsolid formed and was filtered in a sinter-glass funnel, washed withwater, and lyophilized to give a light brown solid (3.2 g). ¹H NMR (300MHz, CDCl₃), δ 8.45 (dd, 2H), 8.05 (m, 2H), 7.9 (d, 2H), 7.8 (dd, 2H),7.6 (dd, 2H), 7.4 (m, 2H), 7.35 (m, 2H), 4.0 (bm, 8H).

Example 4: BT-14 and Salt

As shown in Scheme 4-1, a solution of6-(benzoxazol-2-yl)pyridine-2-carboxylic acid (500 mg) of in DMF (10 mL)was treated with EDC.HCl (1.5 eq), HOBt (1.5 eq), DIPEA (3 eq), andtert-butyl piperazine-1-carboxylate (1.1 eq) at 0° C. The mixture wasallowed to warm to RT for 16 h. After evaporation of the solvent, theresidue was extracted into EtOAc and washed with water. The organiclayer was evaporated under vacuum, crude residue washed with pentanegave light brown solid (120 mg, 48%). ¹H NMR (400 MHz, DMSO-d₆), δ 8.4(d, 1H), 8.2 (t, 1H), 7.9 (t, 2H), 7.8 (d, 1H), 7.5 (dt, 2H), 3.7 (bm,2H), 3.5 (bm, 4H), 3.4 (bm, 2H), 1.4 (s, 9H). LCMS-ES 409.49 [M+H]⁺,431.37 [M+Na]⁺, 447.36 [M+K]⁺.

As shown in Scheme 4-2, the resulting compound from Scheme 4-1 (200 mg)was treated with methanolic HCl (6 mL) at 0° C. The mixture was allowedto warm to RT for 3 h. Evaporation of the solvent and washings withpentane and ether gave of a light brown solid (160 mg, quant.). ¹H NMR(300 MHz, DMSO-d₆), δ 9.30 (bs, 2H), 8.45 (d, 1H), 8.25 (t, 1H), 7.9 (m,3H), 7.5 (quin, 2H), 3.7 (bm, 2H), 3.5 (bm, 2H), 3.3 (bm, 4H), 1.4 (s,9H). LCMS-ES 309.26 [M+H]⁺.

As shown in Scheme 4-3, the resulting salt (25 mg) from Scheme 4-2 wasneutralized with satd. Aq. NaHCO₃ followed by drying in lyophilizer togive 20 mg/96% of BT-14 in hand. The yield was 90%. ¹H NMR (300 MHz,DMSO-d₆), δ 8.4 (d, 1H), 8.2 (t, 1H), 7.90 (t, 2H), 7.75 (d, 1H), 7.5(quin, 2H), 3.95 (bm, 2H), 3.8 (bm, 2H), 3.3 (bm, 2H), 3.2 (bm, 2H);309.37 LCMS-ES [M+H]⁺.

Example 5: BT-15

As shown in Scheme 5-1, 6-(1H-Benzimidazol-2-yl)pyridine-2-carboxylic(50 mg) in DMF (5 mL) was treated with EDC.HCl (1.5 eq), HOBt (1.5 eq),DIPEA (3 eq), and 0.9 eq. of BT-14 HCl salt at 0° C. The mixture wasallowed to warm to RT for 16 h. Filtering over sintered funnel followedby water wash and lyophilizing for moisture removal gave 20 mg of BT-15.¹H-NMR (400 MHz, DMSO-d⁶), δ 12.93 (d, 1H), 8.44 (dd, 1H), 8.36 (t, 1H)8.25 (t, 1H), 8.17 (m, 2H), 7.87 (m, 3H), 7.72 (m, 2H), 7.54 (m, 2H),7.31 (m, 3H), 3.90 (s, 2H), 3.82 (bm, 2H), 3.67 (bm, 2H), 3.58 (bm, 2H).LCMS-ES 530.48 [M+H]+, 265.94 [(M+2H)/2]⁺⁺.

BT-15 has shown LANCL2 binding (FIG. 1A). Its predictive bindingaffinity to LANCL2 is −9.9 and the affinity confirmed by SPR has a Kdvalue of 21.4.

Example 6: BT-13 Salt

As shown in Scheme 6-1, 6-(1H-Benzimidazol-2-yl)pyridine-2-carboxylic(500 mg) in DMF (10 mL) was treated with EDC.HCl (1.5 eq), HOBt (1.5eq), DIPEA (3 eq), and tert-butyl piperazine-1-carboxylate (1.1 eq) at0° C. The mixture was allowed to warm to RT for 16 h. After pouring thereaction mixture into ice-cold water, the precipitate was filtered anddried to give a pale brown solid (600 mg, 70%). TLC (100% ethylacetate). HNMR & LCMS complies. (Yield: 70%). ¹H NMR (300 MHz, DMSO-d₆),δ 12.90 (s, 1H), 8.4 (d, 1H), 8.15 (t, 1H), 7.65 (td, 3H), 7.25 (quin,2H), 3.7 (bm, 2H), 3.5 (bm, 2H), 3.3 (bm, 4H), 1.4 (s, 9H). LCMS-ES408.35 [M+H]⁺.

As shown in Scheme 6-2, the resulting compound from Scheme 6-1 (600 mg)was treated with methanolic HCl (6 mL) for 3 h at 0° C. The mixture wasallowed to gradually warm to RT for 3 hours. Evaporation of the excessmethanolic HCl gave BT-13 HCl (500 mg) as a light brown solid.

Example 7: BT-4 and Salt

As shown in Scheme 7-1, 3-(1H-Benzimidazol-2-yl)benzoic acid (100 mg) inDMF (6 mL) was treated with EDC.HCl (1.5 eq), HOBt (1.5 eq), DIPEA (1eq), and 0.5 eq. of piperazine at 0° C. The mixture was allowed to warmto RT for 16 h. TLC (10% methanol: DCM) shows formation of non-polarspot and absence of starting material. After workup and washings withether 30 mg/95% of BT-4 was isolated. ¹H NMR (400 MHz, DMSO-d₆), δ 13.0(s, 2H), 8.3 (bm, 4H), 7.75 (bm, 4H), 7.60 (bm, 4H), 7.2 (bm, 4H), 3.65(bm, 8H). LCMS-ES 527.36 [M+H]⁺, 264.50 [(M+2H)/2]⁺⁺.

As shown in Scheme 7-2, 30 mg/95% of BT-4 was treated with 4M HCl indioxane for 3 h. Evaporation of the solvent and washing with ether gave10 mg/97% of BT-4 HCl salt. ¹H NMR (400 MHz, DMSO-d₆), δ 8.45 (bm, 4H),7.80 (bm, 8H), 7.50 (bm, 4H), 3.65 (bm, 8H). LCMS-ES 527.44 [M+H]⁺,264.50 [(M+2H)/2]⁺⁺.

Example 8: BT-6 and Salt

As shown in Scheme 8-1, 3-(1H-Benzimidazol-2-yl)benzoic acid (100 mg) inDMF (6 mL) was treated with EDC.HCl (1.5 eq), HOBt (1.5 eq), DIPEA (1eq), and benzene-1,4-diamine (0.5 eq) at 0° C. The mixture was allowedto warm to RT for 16 h. TLC (10% methanol: DCM) shows formation ofnon-polar spot and absence of starting material. After workup andwashings with ether, a light brown solid (60 mg) was isolated. ¹H NMR(300 MHz, DMSO-d₆), δ 13.1 (s, 2H), 10.45 (s, 2H), 8.75 (s, 2H), 8.40(d, 2H), 8.05 (d, 2H), 7.85 (s, 4H), 7.70 (t, 4H), 7.55 (d, 2H) 7.25(quin, 4H). LCMS-ES 549.0 [M+H]⁺ 275.1 [(M+2H)/2]4⁺⁺.

As shown in Scheme 8-2, 60 mg/98% of BT-6 was treated with 4M HCl indioxane for 3 h. After evaporation of the solvent and washed with ethergave 50 mg/96% of BT-6 HCl salt. ¹H NMR (300 MHz, DMSO-d₆), δ 10.60 (s,2H), 9.00 (s, 2H), 8.55 (d, 2H), 8.30 (d, 2H), 7.90 (s, 4H), 7.85 m,6H), 7.50 (m, 4H). LCMS-ES 549.3 [M+H]⁺ 275.3 [(M+2H)/2]⁺⁺.

Example 9: BT-16 and Salt

As shown in Scheme 9-1, 6-(1H-Benzimidazol-2-yl)pyridine-2-carboxylic(100 mg) in DMF (10 mL) was treated with EDC.HCl (1.5 eq), HOBt (1.5eq), DIPEA (3 eq), and benzene-1,4-diamine (0.5 eq) at 0° C. The mixturewas allowed to warm to RT for 16 h. After pouring the reaction mixtureinto ice-cold water, the precipitate was filtered and dried to give apale brown solid (60 mg).

As shown in Scheme 9-2, compound BT-16 (50 mg) was treated with HCl indioxane (3 mL) at 0° C. The mixture was allowed to warm to RT for 4.Evaporation of the excess dioxane HCl gave 30 mg of a brown solid (30mg). ¹H NMR (300 MHz, DMSO-d₆), δ 11.00 (s, 2H), 8.6 (bm, 2H), 8.35 (bm,4H), 8.05 (s, 4H), 7.85 (bm, 4H), 7.40 (bm, 4H). LCMS-ES 551.84 [M+H]⁺.

Example 10: BT-3 and Salt

As shown in Scheme 10-1, 3-(2-Benzoxazolyl)benzoic acid (50 mg) in DMF(10 mL) was treated with EDC.HCl (1.25 eq), HOBt (1.25 eq), DIPEA (1eq), and piperazine (1 eq) at 0° C. The mixture was allowed to warm toRT for 16 h. After diluting the reaction mixture with ice cold water,resulting solid thrown out, filtration, followed by drying gave 30 mg ofBT-3. ¹H NMR (300 MHz, DMSO-d₆), δ 8.2 (bm, 4H), 7.8 (bm, 4H), 7.7 (bm,4H), 7.45 (bm, 4H), 3.6 (bm, 8H). LCMS-ES 529.32 [M+H]⁺.

As shown in Scheme 10-2, BT-3 (30 mg) was treated in methanolic HCl (5mL) at 0° C. The mixture was allowed to warm to RT for 4 h. Afterevaporation of the excess methanolic HCl at vacuum, a brown solid (15mg) formed.

Example 11: BT-5 and Salt

As shown in Scheme 11-1, 3-(2-benzoxazolyl)benzoic acid (50 mg) in DMF(10 mL) was treated with EDC.HCl (1.25 eq), HOBt (1.25 eq), DIPEA (1eq), and benzene-1,4-diamine (0.5 eq) at 0° C. The mixture was allowedto warm to RT for 16 h. Diluting the reaction mixture with ice coldwater, throwing out solids, filtering, followed by drying gave a lightbrown solid (30 mg). ¹H NMR (300 MHz, TFA), δ 9.2 (bs, 2H), 8.8 (bm,2H), 8.6 (bm, 2H), 7.9 (bm, 14H).

As shown in Scheme 11-2, 35 mg of BT-5 was treated in HCl dioxane (5 mL)at 0° C. The mixture was allowed to warm to RT for 4 h. Afterevaporation of the excess dioxane at vacuum, a light brown solid (15 mg)formed. ¹H NMR (300 MHz, TFA), δ 9.3 (bs, 2H), 8.8 (bm, 2H), 8.6 (bm,2H), 7.9 (bm, 14H).

Example 12: BT-17 and Salt

As shown in Scheme 12-1, 6-(Benzoxazol-2-yl)pyridine-2-carboxylic acid(100 mg) in DMF (10 mL) was treated with EDC.HCl (1.5 eq), HOBt (1.5eq), DIPEA (1.2 eq), and benzene-1,4-diamine (0.5 eq) at 0° C. Themixture was allowed to warm to RT for 16 h. Diluting the reactionmixture with ice cold water, throwing out solids, filtering, followed bydrying gave a light brown solid (70 mg). ¹H NMR (400 MHz, TFA), δ 8.85(dd, 4H), 8.55 (t, 2H), 8.1 (bm, 4H), 7.95 (m, 4H), 7.85 (s, 4H).LCMS-ES 553.28 [M+H]⁺.

As shown in Scheme 12-2, BT-17 (60 mg) was treated in dioxane HCl (10mL) at 0° C. to RT for 4 h. After evaporation of the solvent by using alyophiliser, a light brown solid (45 mg) formed. ¹H NMR (400 MHz, TFA),δ 8.90 (bm, 4H), 8.6 (bm, 2H), 8.0 (bm, 10H).

Example 13: BT-ABA-25

The structure of BT-ABA-25 is shown in Scheme 13-1. BT-ABA-25 is aligand of LANCL2 (FIG. 1B). Its predictive binding affinity to LANCL2 is−7.5 and the affinity confirmed by SPR has a Kd value of 1.77e-04.

Example 14: BT-ABA-5a

As shown in Scheme 14-1, a solution of8-vinyl-1,4-dioxaspiro[4.5]decan-8-ol (200 mg, 1 eq) and methyl5-bromofuran-2-carboxylate (1.5 eq) in Et3N (2 mL) was degassed withargon for 10 min. Then, Pd(OAc)2 (0.025 eq), DPPF (0.05 eq) were addedand again degassed for 10 min. The resulting reaction mixture was heatedat 100° C. for 16 h. A light brown solid (130 mg) was isolated by columnchromatography (EtOAx/Hexane 3:7). ¹H NMR (400 MHz, DMSO-d₆), δ 7.30 (d1H), 6.60 (d 1H), 6.45 (dd, 2H), 4.75 (s, 1H), 3.85 (s, 4H), 3.80 (s,3H), 1.85 (m, 2H), 1.65 (m, 2H), 1.50 (m, 4H), LCMS-ES 291.34 [M+H]⁺.

As shown in Scheme 14-2, LiOH (3 eq) was added to a solution of 100 mgof the resulting compound in Scheme 14-1 (compound 4) in THF: H₂O: MeOH(2:1:0.5 mL), and the mixture was stirred at RT for 16 h. The mixturewas then concentrated under reduced pressure, and the crude wasdissolved in minimum amount of water and acidified with 2N HCl up to pH4. Compounds were extracted with EtOAc and concentrated to yield a lightbrown solid (54 mg) which was used for next reaction (Scheme 14-3)without further purification. ¹H NMR (400 MHz, DMSO-d₆), δ 7.50 (d 1H),6.60 (d 1H), 6.45 (dd, 2H), 4.75 (s, 1H), 3.85 (s, 4H), 3.80 (s, 3H),1.85 (m, 2H), 1.65 (m, 2H), 1.50 (m, 4H). LCMS-ES 277.26 [M+H]⁺.

As shown in Scheme 14-3, 3N HCl, 0.1 mL was added to compound 5 (50 mg)in THF at 0° C. with stirring. The mixture was allowed to warm to RT for6 h. TLC shows absence of SM and a non-polar spot. The mixture wasconcentrated under reduced pressure, diluted with water, extracted withEtOAc, and re-concentrated to yield a brown solid (20 mg). ¹H NMR (400MHz, DMSO-d₆), δ 13.00 (bs 1H), 7.20 (d 1H), 6.95 (d 1H), 6.60 (d 1H),6.45 (d, 1H), 6.10 (t, 1H), 3.05 (m, 2H), 2.65 (t, 2H), 2.5, (2H).LCMS-ES 233.21 [M+H]⁺ LCMS-ES 231.27 [M−H]⁻ 463.15 [2M−H]⁻.

Example 15: BT-ABA-6

As shown in Scheme 15-1, a solution of8-vinyl-1,4-dioxaspiro[4.5]decan-8-ol (500 mg, 1 eq), ethyl3-iodobenzoate (0.8 eq), and PPh₃ (0.02 eq) in Et₃N (8 mL) was degassedwith argon for 10 min. Then, Pd(OAc)₂ (0.02 eq) was added and againdegassed for 10 min. The resulting reaction mixture was heated at 95° C.for 16 h. After workup, a pale brown solid (500 mg) was isolated bycolumn chromatography (EtOAc/hexane 3:7). ¹H NMR (400 MHz, DMSO-d₆), δ7.95 (s 1H), 7.80 (d 1H), 7.71 (d 1H), 7.47 (t 1H), 6.65 (d 1H), 6.49(d, 1H), 4.65 (bs 1H), 4.32 (q, 2H), 3.68 (s, 4H), 1.99-1.68 (m, 4H),1.55-1.50 (m, 4H), 1.33 (t 3H). LCMS-ES 315.38 [M−17]⁺.

As shown in Scheme 15-2, a solution of compound 4 (500 mg) inTHF/H₂O/EtOH (4:2:1, 17.5 mL) was cooled to 0° C.; LiOH (2.5 eq) wasadded, and the mixture was stirred while rising to RT over 16 h. Themixture concentrated under reduced pressure, and the crude was dissolvedin minimum amount of water and acidified with 1N HCl up to pH 3-4.Purification by column chromatography (EtOAc/hexane 1:1) gave a paleyellow solid (220 mg). ¹H NMR (400 MHz, DMSO-d₆), δ 13.00 (bs 1H), 7.95(s 1H), 7.78 (d 1H), 7.67 (d 1H), 7.44 (t 1H), 6.64 (d 1H), 6.48 (d,1H), 4.65 (s 1H), 3.86 (s, 4H), 1.87-1.61 (m, 4H), 1.55-1.50 (m, 4H).LCMS-ES 287.34 [M−17]⁺.

As shown in Scheme 15-3, 2N HCl (1.5 mL) was added to a mixture of 100mg of compound 5 (100 mg) in THF at 0° C. with stirring. The mixture wasallowed to warm to RT for 6 h. The solution was then concentrated underreduced pressure, diluted with water, extracted with EtOAc, andre-concentrated to get a pale yellow solid (20 mg). ¹H NMR (400 MHz,DMSO-d₆), δ 13.00 (bs 1H), 8.00 (s, 1H), 7.80 (d 1H), 7.65 (d 1H), 7.45(t 1H), 6.75 (d 1H), 6.45 (d, 1H), 6.10 (t, 1H), 5.15 (s 1H), 2.65 (m,2H), 2.15 (m, 2H), 1.90 (m, 4H), LCMS-ES 259.37 [M−H]⁻ 519.48 [2M−H]⁻.

Example 16: BT-ABA-13

As shown in Scheme 16-1, dihydropyran (1.3 eq) and TsOH (0.1 eq) wasadded to a solution of compound 2 (2.5 g, 1 eq) in CH₂Cl₂ (50 mL) at 0°C. with stirring. The resulting solution was allowed to gradually warmto RT for 14 h. A pale yellow liquid was isolated by columnchromatography (EtOAc/hexane 1:9). The compound was used in the nextstep without further purification.

As shown in Scheme 16-2, compound 3 (2.5 g, 1.0 eq),4,4,5,5-tetramethyl-[1,3,2]dioxaborolane (1.2 eq), andbis(cyclopentadienyl)zirconium chloride hydride (0.15 eq) were added toEt₃N. The resulting reaction mixture was heated at 60-70° C. for 16 h.The reaction mixture was diluted with hexanes. The precipitate wasremoved by filtration over short pad of silica gel and washed withhexanes. Upon concentration of the hexane solutions, a colorless oilyliquid (1.3 g) was obtained. ¹H NMR (400 MHz, CDCl3), δ 6.60 (d 1H),5.60 (d 1H), 6.35 (d, 1H), 4.75 (s, 1H), 3.85 (s, 3H), 2.80 (m, 2H),2.35 (m, 2H), 2.05 (m, 4H).

As shown in Scheme 16-3, a solution of compound 4 (550 mg, 1.1 eq),methyl 6-bromopicolinate (1.0 eq), K₂CO₃ (2.0 eq) in mixture of DME/H₂O9:1 (8 mL) was degassed with argon for 10 min. Then, Pd[(P(Ph)₃]₄ (0.04eq) was added. The resulting reaction mixture was heated at 100° C. for16 h. Concentration of the reaction solution followed by columnchromatography (EtOAc/hexane 1:3) yielded a pale yellow solid (230 mg).LCMS-ES 404.39 [M+H]⁺, 302.26 [M−101]⁺.

As shown in Scheme 16-5, TsOH (0.1 eq) was added to a solution ofcompound 5 (230 mg, 1.0 eq) in acetone/H₂O 1:1 (6 mL). The resultingreaction mixture was stirred at room temperature for 16 h. Concentrationof the reaction mixture followed by column chromatography (EtOAc/hexane7:3) gave a pale yellow liquid (110 mg). ¹H NMR (400 MHz, CDCl3), δ 8.00(d 1H), 7.80 (t, 1H), 7.50 (d, 1H), 6.90 (m 2H), 4.00 (s, 3H), 2.80 (m,2H), 2.35 (m, 2H), 2.10 (m, 4H), LCMS-ES 276.38 [M+H]⁺.

As shown in Scheme 16-6, LiOH (2.5 eq) was added to a solution ofcompound 6 (75 mg) in THF/H₂O 3:1 (3 mL) 0° C. with stirring. Themixture was allowed to warm to RT for 6 h. The reaction mixture wasacidified with citric acid and extracted with a mixture of THF andEtOAc. Concentration of the organic solution gave an off-white solid (10mg). ¹H NMR (300 MHz, DMSO-d₆), δ 13.05 (bs, 1H), 7.90 (m, 2H), 7.65 (d,1H), 7.05 (d, 1H), 6.80 (d, 1H), 5.20 (s, 1H), 2.65 (m, 2H), 2.20 (bd2H), 2.10-1.90 (m, 4H), LCMS-ES 262.27 [M+H]⁺.

Example 17: BT-ABA-16

As shown in Scheme 17-1, a solution of compound 4 (437 mg, 1.2 eq),methyl 2-bromoisonicotinate (1.0 eq), K₂CO₃ (2.0 eq) in mixture ofDME/H₂O 9:1 (8 mL) was degassed with argon for 10 min. Then,Pd[(P(Ph)₃]₄ (0.04 eq) was added. The resulting reaction mixture washeated at 90° C. for 12 h. Concentration of the reaction solutionfollowed by column chromatography (EtOAc/hexane 1:3) yielded a paleyellow liquid (300 mg). ¹H NMR (300 MHz, CDCl₃), δ 8.70 (d, 1H), 7.85(s, 1H), 7.65 (d, 1H), 6.85 (d, 1H), 6.65 (d, 1H), 4.70 (m, 1H), 3.95(m, 4H), 2.20-1.40 (m, 16H), LCMS-ES 404.54 [M+H]⁺, 302.53 [M−101]⁺.

As shown in Scheme 17-2, TsOH (0.1 eq) was added to a solution of 5 (300mg, 1.0 eq) in acetone/H₂O 1:1 (6 mL). The resulting reaction mixturewas stirred at RT for 48 h. Concentration of reaction mixture followedby column chromatography (EtOAc/hexane 7:3) gave an off-white solid (160mg). ¹H NMR (300 MHz, DMSO-d6), δ 8.70 (d, 1H), 7.85 (s, 1H), 7.65 (d,1H), 7.05 (d, 1H), 6.85 (d, 1H), 5.20 (s, 1H), 3.90 (s, 3H), 2.65 (td,2H), 2.15 (bd, 2H), 2.00 (m, 2H), 1.85 (m, 2H), LCMS-ES 276.22 [M+H]⁺.

As shown in Scheme 17-3, LiOH (2.5 eq) was added to a solution ofcompound 6 (100 mg) in THF/H₂O 3:1 (3 mL) at 0° C. with stirring. Themixture was allowed to warm to RT for 16 h. The reaction mixture wasacidified with citric acid and extracted with mixture of THF and EtOAc.Concentration under reduced pressure gave an off-white solid (20 mg). ¹HNMR (300 MHz, DMSO-d6), δ 13.60 (bs, 1H), 8.70 (d, 1H), 7.85 (s, 1H),7.60 (d, 1H), 7.00 (d, 1H), 6.85 (d, 1H), 5.20 (s, 1H), 2.65 (m, 2H),2.20-1.80 (m, 6H), LCMS-ES 262.28 [M+H]⁺.

Example 18: BT-ABA-14

As shown in Scheme 18-1, a solution of compound 4 (300 mg, 1.2 eq),methyl 4-bromopicolinate (1.0 eq), K₂CO₃ (2.0 eq) in mixture of DME/H₂O9:1 (8 mL) was degassed with argon for 10 min. Then, Pd[(P(Ph)₃]₄ (0.04eq) was added. The resulting reaction mixture was heated at 90° C. for12 h. Concentration of the reaction solution followed by columnchromatography (EtOAc/hexane 1:3) yielded a pale yellow liquid (200 mg).¹H NMR (300 MHz, CDCl₃), δ 8.50 (d, 1H), 8.20 (bs, 1H), 7.45 (d, 1H),6.70 (d, 1H), 6.50 (d, 1H), 4.60 (m, 1H), 3.95 (m, 4H), 2.20-1.40 (m,16H), LCMS-ES 390.35 [M+H]⁺.

As shown in Scheme 18-2, TsOH (0.1 eq) was added to a solution of 5 (200mg, 1.0 eq) in acetone/H₂O 1:1 (6 mL). The resulting reaction mixturewas stirred at room temperature for 48 h. The reaction mixture wasacidified with citric acid and extracted with mixture of THF and EtOAc.The solution was concentrated to give an off-white solid (18 mg). ¹H NMR(300 MHz, DMSO-d₆), δ 8.60 (d, 1H), 8.05 (s, 1H), 7.60 (d, 1H), 6.90 (d,1H), 6.70 (d, 1H), 5.20 (bs, 1H), 2.65 (m, 2H), 2.15 (bd, 2H), 2.05-1.80(m, 4H), LCMS-ES 262.27 [M+H]⁺.

RECEPTOR BINDING EXAMPLES Example 19: LANCL2 Binding Example

Computational modeling studies and biochemical validation were combinedto guide the selection on compounds that bind to LANCL2. Latestiterations of surface plasmon resonance (SPR) technology provide an invitro, high throughput, quantitative means to determine molecularinteraction between label-free proteins and small molecules (>25 Da) inreal time. BIACORE™ T200 (GE Healthcare, Piscataway, N.J.) technologyfurther provides an added benefit of GMP/GLP compliance and autonomouslarge-scale data acquisition either of screens or detailed titrations inless than 24-hour period. Molecular interactions of interest areroutinely validated by BIACORE™ T200 SPR technology.

Methods

High-Throughput Screening Via Molecular Modeling of LANCL2-CompoundInteractions.

Auto-Doc Vina [14] is a state of the art software suite capable ofhigh-throughput parallel computations to ascertain LANCL2-botanicalcompound binding. The software suite first computes (i) the forces offree energy associated with the bound complex and subsequently (ii) theconformational space available for the complex formation between targetand ligand. These methods are stochastic in nature therefore requirerepeated independent screens to exhaustively search all parameter spacesand provide confidence in predictions. Currently the model of LANCL2 isavailable through homology modeling of LANCL1 [15]. AutoDockTools, thegraphical front-end for AutoDock and AutoGrid, was used to define thesearch space, including grid box center and x,y,z-dimensions [16].AutoDock Vina generated five bound conformations for each compound. Thedocking is applied to the whole protein target, with a grid covering thewhole surface of the protein. Docking log files were generatedconsisting of binding energies of each predicted binding mode for allthe compounds for all surfaces.

Kinetic Determination of LANCL2-Small Molecule Interaction.

BIACORE™ T200 was used to determine the kinetic parameters for thebinding of small molecules BT-11, BT-ABA-5a, BT-6, and BT-15 (analytes)to LANCL2 (ligand). Data were generated in a dose dependent (5-8titration points) manner in triplicate, and analyzed to determinebinding model (Langmuir, conformational shift, etc.), real timeassociated and disassociation constants, and equilibrium dissociationconstant. SPR technology allowed validation of specificLANCL2-phytochemical interactions as well as to gain gold-standardinsight into mechanism and rate of binding. The experiments wereperformed on carboxymethyldextran (CM5) sensor chips by covalentlyattaching LANCL2 to by amine coupling. Flow cells 1 and 2 of the sensorchip were activated for 720 sec at 10 μl/min with of 1:1 mixture of 0.1M N-hydroxysuccinimide (NHS) and 0.5 M1-ethyl-3-(-3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC).Stock LANCL2 (0.41 mg/mL) was diluted to 8.2 μg/mL (1:50 dilution) in 10mM sodium acetate, pH 5.0 and injected over the activated flow cell 2surface for 1000 sec at a flow rate of 10 μl/min. After the capture ofLANCL2 on flow cell 2 (11000 RU), surfaces of flow cells 1 and 2 weredeactivated by injecting 1M ethanolamine for 720 sec at 10 μl/min. Therunning buffer was 25 mM MOPS containing 0.05% T-20 and 0.15 M NaCl, pH6.5. Kinetic studies were performed by injecting differentconcentrations of the BT-11 (25 μM, 12.5 μM, 6.25 μM, 3.13 μM, 1.56 μM,and 0.76 μM), BT-ABA-5a (40 μM, 20 μM, 10 μM, 5 μM, 2.5 μM, and 1.25 μM)and BT-15/BT-6 (20 μM, 10 μM, 5 μM, 2.5 μM, 1.25 μM, 0.625 μM, and 0.313μM) in triplicates. Each sample was injected for 60 sec (contact time)followed by a dissociation time of 60 sec at a flow rate of 100 μL/min.A stabilization time of 180 sec was used before the next injection. Datawas analyzed with BIACORE™ T200 Evaluation Software (version 1) todetermine the affinity binding constant (KD) using a 1:1 binding model.

Results

Both BT-11 and BT-15 Strongly Bind to LANCL2.

In order to confirm binding of BT-11 and BT-15 to the LANCL2 protein, weperformed SPR analyses in a BIACORE™ T-200 instrument. SPR, an opticaltechnique utilized for detecting molecular interactions, was used tomeasure binding affinity between LANCL2 and its ligands (i.e., BT-11 andBT-15). We immobilized purified recombinant LANCL2 protein on BIACORE™sensor chips and injected small molecules over the protein surface usingthe microfluidic system of the instrument. Changes in the total mass onchip surface were measured, which corresponds to the small binding tothe protein. By injecting a series of small molecule concentrations wewere able to calculate steady state binding affinities for BT-11 bindingto LANCL2 and BT-15 binding to LANCL2. Binding sensorgrams showed atypical small molecule protein interaction with very fast on rates andvery fast off rates (FIG. 8, panels A and C). These fast interactionsare beyond the technical abilities of the instrument. Therefore,reliable association rate constant (k_(a)) and dissociation rateconstant (k_(d)) were not determined. The equilibrium dissociationconstant (K_(D)) is commonly used to describe the affinity between aligand and a protein, such as how tightly a ligand binds to a particularprotein. Ligand-protein affinities are influenced by non-covalentintermolecular interactions between the two molecules such as hydrogenbonding, electrostatic interactions, hydrophobic and Van der Waalsforces. By plotting the equilibrium binding level against the compoundconcentration, we were able to measure the steady state affinity (K_(D))for each interaction (FIG. 8, panels B and D). Both small moleculesshowed a similar binding affinity for LANCL2 (BT-11: 7.7 uM, BT-15 11.4uM).

BT-ABA-5a and BT-6 Strongly Bind to LANCL2.

Similar to the results described above and in order to confirm bindingof BT-6 and BT-ABA-5a to LANCL2, we performed SPR analyses in a BIACORE™T-200 instrument. In this case we also immobilized purified recombinantLANCL2 protein on BIACORE™ sensor chips and injected small moleculesover the protein surface using the microfluidic system of theinstrument. Changes in the total mass on chip surface were measured,which corresponds to the small binding to the protein. Taking a closerlook at the binding sensorgrams (FIGS. 9A and 9B), our results show howBT-6 and BT-ABA-5a are very fast to bind but not as fast as off, incomparison to BT-11/BT-15, which are very fast on and very fast off. Ofnote, the occupancy time for BT-ABA-5a shows the slowest off rate,meaning that BT-ABA-5a stays the longest in the binding pocket ofLANCL2. This longer binding can potentially impact the activation of theLANCL2 pathway by triggering more efficacious anti-inflammatory andanti-diabetic and other therapeutic responses.

Other compounds have been tested via SPR, and the results arecomprehensibly shown in FIGS. 1A and 1B.

EXPERIMENTAL STUDIES EXAMPLES Example 20: Use of BT-11 on an Acute Modelof IBD Introduction

Inflammatory bowel disease (IBD), a chronic, recurring disease of thegastrointestinal tract, afflicts over 1.4 million people in the U.S. IBDcomprises two different manifestations: ulcerative colitis and Crohn'sdisease. Current therapies against IBD are modestly successful and havesignificant adverse side effects for the long-term management of thedisease [17]. Whereas Crohn's disease represents the chronic stage ofthe disease, acute ulcerative colitis (UC) is manifested as an earlypathology that affects the colonic tissue. UC is a chronic idiopathicinflammatory disorder of the GI tract characterized by mucosalinflammation of the rectum that extends proximally through the colon, ina continuous fashion, but to a variable extent. The disorder ischaracterized by a relapsing and remitting course of variable severity.The majority of patients present with left-sided or distal disease ofmild-to-moderate severity. Most remain in remission for long periodswith maintenance medical therapy. However, natural history studiessuggest that between 10 and 40% will undergo a colectomy at some pointduring the course of their disease.

Medical treatment of steroid-refractory severe UC has expanded somewhatin recent years with the availability of both ciclosporin and infliximabas rescue agents; however surgery still remains the only “curative”option. The present invention provides a novel drug product for thetreatment of UC by targeting a novel receptor named LANCL2. BT-11, ourtop lead compound, is administered orally and distributed systemically,and exerts immune modulatory effects in UC by targeting LANCL2 in gutimmune cells. Our pre-clinical efficacy studies in acute UC in miceshowed how administration with BT-11 reduces the disease activity indexand improves gut inflammation by significantly decreasing leukocyticinfiltration in the gut mucosa, as well as decreasing mucosal thickeningand epithelial erosion. Gene expression analyses confirmed that oraladministration of BT-11 upregulates the expression of IL-10 and LANCL2,and downregulates the expression of TNFαmRNA in a model of acuteDSS-induced ulcerative colitis in mice.

Methods

Mice.

C57BL/6 were purchased from the Jackson Laboratory and housed underspecific pathogen-free conditions in ventilated racks. LANCL2−/− micewere purchased from the KOMP repository at University of CaliforniaDavis. All mice were maintained in animal facilities. All experimentalprotocols were approved by an institutional animal care and usecommittee and met or exceeded guidelines of the National Institutes ofHealth Office of Laboratory Animal Welfare and Public Health Servicepolicy.

DSS-Induced Colitis.

Colitis was induced in C57BL/6J mice by administration of 5% (w/v)dextran sodium sulfate (DSS; molecular weight 42 kDa; ICN Biochemicals,Aurora, Ohio) added to the drinking water. Colonic inflammation wasassessed 7 days after DSS treatment. The groups in the DSS projectconsisted of i. non-DSS vehicle-treated mice, ii. non-DSS, BT-11 (80mg/Kg) treated mice, iii. DSS-treated, vehicle-treated mice, and iv.DSS-treated, BT-11 (80 mg/Kg) treated mice. Twelve mice were included ineach group.

Histopathology.

Colonic sections from IBD studies in mice were fixed in 10% bufferedneutral formalin, later embedded in paraffin and then sectioned (5 μm)and stained with H&E stain for histological examination. Colons weregraded with a compounded histological score including the extent of (1)leukocyte infiltration, (2) mucosal thickening and (3) epithelial cellerosion. The sections were graded with a score of 0-4 for each of theprevious categories, and data were analyzed as a normalized compoundedscore.

Quantitative Real-Time PCR.

Total RNA was isolated from mouse colons using an RNEASY PLUS MINI KIT(Qiagen, Valencia, Calif.) according to the manufacturer's instructions.Total RNA (1 μg) was used to generate a cDNA template using an ISCRIPT™cDNA Synthesis kit (Bio-Rad, Hercules, Calif.). The total reactionvolume was 20 μL, with the reaction incubated as follows in an MJ MINI™thermal cycler (Bio-Rad): 5 min at 25° C., 30 min at 52° C., 5 min at85° C., and hold at 4° C. PCR was performed on the cDNA using Taq DNApolymerase (Life Technologies, Carlsbad, Calif.). Each gene amplicon waspurified with the MINELUTE PCR Purification kit (Qiagen) and quantifiedboth on an agarose gel by using a DNA mass ladder (Promega, Madison,Wis.) and with a nanodrop. These purified amplicons were used tooptimize real-time PCR conditions and to generate standard curves in thereal-time PCR assay. Primers were designed using Oligo 6 software.Primer concentrations and annealing temperatures were optimized for theICYCLER IQ™ system (Bio-Rad) for each set of primers using the system'sgradient protocol. PCR efficiencies were maintained between 92 and 105%and correlation coefficients >0.98 for each primer set duringoptimization and also during the real-time PCR of sample DNA. cDNAconcentrations for genes of interest were examined by real-time qPCRusing an ICYCLER IQ™ System and the IQ™ SYBR® Green Supermix (Bio-Rad).A standard curve was generated for each gene using 10-fold dilutions ofpurified amplicons starting at 5 pg of cDNA and used later to calculatethe starting amount of target cDNA in the unknown samples. SYBR® green Iis a general double-stranded DNA intercalating dye and may thereforedetect nonspecific products and primer/dimers in addition to theamplicon of interest. To determine the number of products synthesizedduring the real-time PCR, a melting curve analysis was performed on eachproduct. Real-time PCR was used to measure the starting amount ofnucleic acid of each unknown sample of cDNA on the same 96-well plate.

Statistical Analysis.

Parametric data were analyzed using the ANOVA followed by Scheffe'smultiple comparison method. Nonparametric data were analyzed by usingthe Mann-Whitney's U test followed by a Dunn's multiple comparisonstest. ANOVA was performed by using the general linear model procedure ofSAS, release 6.0.3 (SAS Institute). Statistical significance wasassessed at a P<0.05.

Results

BT-11 Improves Disease and Tissue Pathology in a DSS Model of Colitis.

The objective of this study was to investigate whether administration ofBT-11 activates LANCL2 and exerts anti-inflammatory properties in thecontext of IBD. To assess the efficacy of our exemplary compound BT-11in an acute model of IBD, we treated C57BL/6J mice with 5% DSS on a7-day challenge. Throughout the challenge period, the treatment withBT-11 significantly improved the score in disease activity (FIG. 10,panel A). Furthermore, the macroscopic lesions in the spleen (FIG. 10,panel B), the MLNs (FIG. 10, panel C) and the colon (FIG. 10, panel D)were also significantly decreased following activation of the LANCL2pathway by using BT-11 at day 7 post-challenge.

BT-11 Improves Colonic Histopathology in Mice with Acute InflammatoryColitis in a Dose Response Manner.

We next examined the effect of BT-11 on histopathological colonicinflammatory lesions. In line with our observations of disease activityand gross lesions, histopathological analyses confirmed that treatmentwith BT-11 significantly decreased by 5 times the inflammation in thegut mucosa based on assessment of leukocytic infiltration (FIG. 11,panel G), epithelial erosion (FIG. 11, panel H), and mucosal thickening(FIG. 11, panel I). Representative colonic micrographs show howtreatment with BT-11 during DSS-induced colitis in mice significantlyimproves the status of the gut mucosa by improving epithelial cellintegrity and reducing the destruction of the gut architecture, as wellas the infiltration of several immune subsets (FIG. 11, panels A-F). Weperformed dose-response studies with BT-11 and we interestingly observedhow the three hallmarks of colonic inflammation (leukocyticinfiltration, mucosal thickening, and epithelial erosion) were decreasedin mice with colitis as the dose of BT-11 was increased from 10 to 80mg/Kg (FIG. 12, panels A-C).

Oral Treatment with BT-11 Reduces the Expression of TNFα and UpregulatesLANCL2 and IL-O.

To more closely investigate the effect of BT-11 on the modulation of theimmune system, we assessed genetic expression of IL-10, LANCL2, andTNFα. Results show how treatment with BT-11 down-regulated theexpression of tumor necropsis factor alpha (TNFα) (FIG. 13, panel A), aswell as upregulated the levels of Interleukin 10 (IL-10) (FIG. 13, panelB) and the LANCL2 receptor (FIG. 13, panel C), therefore creating apositive feedback loop that promotes anti-inflammatory effects anddown-regulates the inflammatory response driven by TNFα. By performing adose-response study we could hypothesize that our ligand BT-11 and thefollowing activation of the LANCL2 pathway directly increases theproduction of colonic IL-10, as its expression assessed by flowcytometry follows dose-response dynamics with BT-11 (FIG. 14, panel B).We observed that the reduction of colonic TNFα expressing cells wassignificantly different at both 40 and 80 mg/Kg of BT-11, but not onlower doses, such as 10 or 20 mg/Kg (FIG. 14, panel A). We also observedhow FOXP3 expression in the MLN is dose-dependent (FIG. 14, panel C).

The Effects of BT-11 During Acute Colitis are Dependent on LANCL2.

In order to demonstrate how the beneficial effects of administrationwith BT-11 are exerted during acute colitis in mice, we performedstudies comparing such effects in wild-type and LANCL2 knock-out(LANCL2−/−) mice. Our results demonstrate that LANCL2 is necessary forBT-11 to exert its anti-inflammatory benefits, as the loss of LANCL2prevented the mice to recover from acute DSS-induced colitis (FIG. 15,panel A). Likewise, the loss of LANCL2 abrogated the decrease inmacroscopic score in the colon (FIG. 15, panel B), the MLN (FIG. 15,panel C), and the spleen (FIG. 15, panel D) when comparing wild-type andLANCL2−/− littermates. Furthermore, the effect of BT-11 in lesionformation in the colonic mucosa is also LANCL2-dependent, as we assessedhistopathological analyses in LANCL2−/− mice treated with either vehicleor BT-11 and we observed how the loss of LANCL2 completely abrogates theeffect of BT-11 (FIG. 16).

To further characterize the cellular responses following treatment withBT-11, we performed further LANCL2 knockout studies to determine if thedecrease of pro-inflammatory proteins and the increase ofanti-inflammatory factors were ablated. Our flow cytometry resultsdemonstrate that the reduction of the pro-inflammatory factor MCP1 isLANCL2-dependent in both the colon (FIG. 17, panel A) and the MLN (FIG.17, panel B), since the loss of the LANCL2 gene abrogates the effect ofBT-11. We also found that the secretion of TNFα in the colon isLANCL2-dependent (FIG. 17, panel C) as well as the upregulation ofMHC-II+CD11c+ populations of granulocytes (FIG. 17, panel D). In linewith these results, we found that the upregulation of IL-10 secretionafter BT-11 treatment is completely abrogated in LANCL2 knockout mice inboth the colon (FIG. 17, panel E) and the spleen (FIG. 17, panel F),showing, once again, the dependency of our top lead compound with ourtarget of interest.

Discussion

LANCL2 has emerged as a novel therapeutic target for inflammatory andimmune-mediated diseases [18]. Our in vivo results demonstrate for thefirst time that oral treatment with LANCL2 ligand BT-11 ameliorates gutimmunopathology in mouse models of IBD by suppressing inflammation.LANCL2 has received some recent attention as a potential therapeutictarget due to its function related to ABA binding and signaling [19] andthe recent discovery of an alternative membrane-based mechanism of PPARγ activation [8]. Furthermore, we determined the LANCL2 expression in aseries of mouse tissues, which showed that beside brain and testis,LANCL2 is also expressed in other tissues, such as thymus, spleen,colon, and Peyer's patches, which indicates the possible relationshipbetween LANCL2 and immune responses and suggest the broader potential ofLANCL2 as a therapeutic target.

Previously, we have reported that ABA transactivates PPARγ in vitro andsuppresses systemic inflammation similar to other PPAR γ agonists. Sinceboth ABA and NSC61610 target LANCL2, NSC61610 might also act via PPAR γactivation. Experimental results show that NSC61610 treatment activatesPPAR γ in raw macrophages, thereby providing evidence of a potentialsignaling relationship between LANCL2 and PPAR γ and indicating thatNSC61610 might target the LANCL2-PPAR γ axis in vitro. To investigatethe importance of LANCL2 in NSC61610-mediated activation of PPAR γ, wedetermined whether knocking down LANCL2 in raw macrophages by usingsiRNA impaired or abrogated the effect of NSC61610 on PPAR γ reporteractivity. Our findings indicate that knocking down LANCL2 significantlyattenuates the effect of NSC61610 on PPAR γ activity [12]. In thisexample, we demonstrate how the administration of BT-11 exertsanti-inflammatory properties by decreasing not only the score in diseaseactivity index and the macroscopic scores in spleen, MLN, and colon(FIG. 10) but also significantly reducing histopathological lesions(FIG. 11). We demonstrated how these two specific effects were dependenton LANCL2 (FIG. 15 and FIG. 16). We also demonstrated that BT-11 reducesthe levels of TNFα and upregulates both LANCL2 and IL-10 (FIG. 13). Wealso demonstrated that there effects are LANCL2-dependent as we did notobserve these trends in LANCL2−/− mice (FIG. 17). These results confirmthat LANCL2 is a novel therapeutic target for inflammatory diseases andBT-11 is a compound that targets it.

Example 21: Use of BT-11 on a Chronic Model of Crohn's DiseaseIntroduction

As stated above, inflammatory bowel disease (IBD), with its two clinicalmanifestations, ulcerative colitis and Crohn's disease, is animmune-mediated disease characterized by widespread inflammation andimmune cell infiltration of the gastrointestinal tract. The etiology ofIBD is multifactorial, and entails interaction among geneticpredisposition, environmental factors, and the gut microbiota.

The present example will focus on the chronic manifestation of IBD:Crohn's disease. Whereas the inflammation in ulcerative colitis ischaracterized by a continuous pattern that involves the superficialmucosal and submucosal layers but is limited to the colon, in Crohndisease this inflammation is transmural and discontinuous, and anyregion of the gut can be affected beyond the ileum, which is mostaffected. Crohn's disease pathogenesis is complex and influenced bygenetic and environmental factors and immune-mediated injury to the gutmucosa brought about by prolonged activation of the mucosal immunesystem.

Treatments targeted to downmodulate the immune and inflammatoryresponses, such as the corticosteroid prednisone or the anti-tumornecrosis factor-α antibody REMICADE® (Janssen Biotech, Inc., Horsham,Pa.) (infliximab), have shown promise in reducing severity andreoccurrence of the disease. These treatments, however, are alsoassociated with various adverse side effects, such as cushingoidappearance, weight gain, and systemic immunosuppression, thus stressingthe need to develop safer alternatives for the long-term management ofIBD [20].

The present invention provides a novel drug product for the treatment ofCrohn's disease by targeting a novel receptor named LANCL2. BT-11, anexemplary compound, is administered orally and distributed systemically,and exerts immune modulatory effects in not only UC but also Crohn'sdisease by targeting LANCL2 in gut immune cells. Our pre-clinicalefficacy studies in chronic models of Crohn's disease in mice showed howadministration with BT-11 reduces the disease activity index andimproves gut inflammation by significantly decreasing leukocyticinfiltration in the gut mucosa, as well as decreasing mucosal thickeningand epithelial erosion. Gene expression analyses confirmed that oraladministration of BT-11 upregulates the expression of LANCL2, anddownregulates the expression of TNFα mRNA in a chronic model of IBD inmice. Furthermore, the administration of BT-11 reduces proinflammatorymacrophages and dendritic cell infiltration into the colonic laminapropria as well as upregulated FOXP3-expressing CD4+ T cells anddownregulated the number of effector Th1 cells in the colon. We alsoperformed knock-out studies to confirm that these effects areLANCL2-dependent. Finally, in the induction sites, BT-11 is capable ofdownregulating the generation of Th17 cells as well as upregulating theregulatory CD4+ T cell compartment via upregulation of FOXP3 expression.

Methods

Mice.

C57BL/6 and IL-10 knockout mice were purchased from the JacksonLaboratory and housed under specific pathogen-free conditions inventilated racks. LANCL2−/− mice were purchased from the KOMP repositoryat University of California Davis. All mice were maintained in animalfacilities. All experimental protocols were approved by an institutionalanimal care and use committee and met or exceeded guidelines of theNational Institutes of Health Office of Laboratory Animal Welfare andPublic Health Service policy.

CD4+ T Cell Enrichment and Sorting.

Splenocytes obtained from C57BL/6J (wild-type) mice were enriched inCD4+ T cells by magnetic negative sorting using the I-Mag cellseparation system (BD Pharmingen). Cells were incubated with a mixtureof biotinylated Abs followed by a second incubation with streptavidinparticles and exposed to a magnet to remove unwanted cells. The purityof the CD4+-enriched cell suspension was between 93 and 96%.CD4-enriched cells were used for adoptive transfer, or further purifiedby FACS. For FACS sorting, cells were labeled with CD45RB, CD4, and CD25and separated into CD4+ CD45RBhigh CD25− cells (i.e., effector T cells)in a FACSARIA™ cell sorter (BD Biosciences, San Jose, Calif.). Thepurity of the FACS-sorted CD4+ subsets was ≥98%.

Adoptive Transfer.

Six-week-old SCID and RAG2−/− mice were administered intraperitoneally(i.p.) 4×10⁵ CD4+ CD45RBhigh CD25− from C57BL/6J (wild-type) orLANCL2−/− mice. Mice were weighed on a weekly basis and clinical signsof disease were recorded daily for 14 wk. Mice that developed severesigns of wasting disease were sacrificed. Otherwise, mice weresacrificed 90 days after transfer. The groups for adoptive transferstudies went as follows: i. non-transferred vehicle treated, ii.Non-transferred BT-11 (80 mg/Kg) treated, iii. Transferred vehicletreated, iv. Transferred BT-11 (80 mg/Kg) treated. 12 mice were used ineach group.

Histopathology.

Colonic sections from IBD studies in mice were fixed in 10% bufferedneutral formalin, later embedded in paraffin and then sectioned (5 am)and stained with H&E stain for histological examination. Colons weregraded with a compounded histological score including the extent of (1)leukocyte infiltration, (2) mucosal thickening and (3) epithelial cellerosion. The sections were graded with a score of 0-4 for each of theprevious categories, and data were analyzed as a normalized compoundedscore.

Cell Isolation.

Spleens and mesenteric lymph nodes (MLN) were excised and crushed in1×PBS/5% FBS using the frosted ends of two sterile microscope slides.Single cell suspensions were centrifuged at 300×g for 10 min and washedonce with 1×PBS. Red blood cells were removed by osmotic lysis prior tothe washing step. All cell pellets were resuspended in FACS buffer(1×PBS supplemented with 5% FBS and 0.09% sodium azide) and subjected toflow cytometric analysis. Paralelly, colons were excised and laminapropria leukocytes (LPL) were isolated. Tissue pieces were washed in CMF(1×HBSS/10% FBS/25 mM Hepes), and tissue was incubated twice with CMF/5mM EDTA for 15 min at 37° C. while stirring. After washing with 1×PBS,tissue was further digested in CMF supplemented with 300 U/ml type VIIIcollagenase and 50 U/ml DNAse I (both Sigma-Aldrich) for 1.5 hs at 37°C. while stirring. After filtering the supernatants, cells were washedonce in 1×PBS, pellets were resuspended in FACS buffer and subjected toflow cytometric analysis.

Immunophenotyping and Cytokine Analysis by Flow Cytometry.

For fluorescent staining of immune cell subsets 4-6×10⁵ cells wereincubated for 20 min with fluorochrome-conjugated primary mouse specificantibodies: anti-CD3 PE-Cy5 clone 145-2C11 (eBioscience, San Diego,Calif.), anti-CD4 PE-Cy7 clone GK1.5 (eBioscience), anti-CD4 APC cloneRM4-5 and anti-CD25 Biotin clone 7D4 (BD Biosciences). Cells were washedwith FACS buffer (1×PBS supplemented with 5% FBS and 0.09% sodiumazide). For intracellular staining of transcription factors andcytokines, cells were fixed and permeabilized using a commercial kitaccording to the manufacturer's instructions (eBioscience). Briefly,cells were fixed and permeabilized for 20 minutes, Fc receptors wereblocked with mouse anti-CD16/CD32 FcBlock (BD Biosciences) and cellswere stained with fluorochrome-conjugated antibodies towards anti-mouse,FOXP3 FITC clone FJK-16s, anti-mouse ROR gamma (t) PE, clone B2B andanti-mouse IL17-A APC, clone eBio17B7 (eBioscience). All samples werestored fixed at 4° C. in the dark until acquisition on a FACS Aria flowcytometer (BD Biosciences). A live cell gate (FSC-A, SSC-A) was appliedto all samples followed by single cell gating (FSC-H, FSC-W) beforecells were analyzed for the expression of specific markers. Dataanalysis was performed with FACS DIVA™ (BD Biosciences) and Flow Jo(Tree Star Inc.).

Quantitative Real-Time PCR.

Total RNA was isolated from mouse colons using a RNEASY PLUS MINI KIT(Qiagen) according to the manufacturer's instructions. Total RNA (1 jag)was used to generate a cDNA template using an ISCRIPT™ cDNA Synthesiskit (Bio-Rad). The total reaction volume was 20 μL, with the reactionincubated as follows in an MJ MINI™ thermal cycler (Bio-Rad): 5 min at25° C., 30 min at 52° C., 5 min at 85° C., and hold at 4° C. PCR wasperformed on the cDNA using Taq DNA polymerase (Invitrogen). Each geneamplicon was purified with the MINELUTE PCR Purification kit (Qiagen)and quantified both on an agarose gel by using a DNA mass ladder(Promega) and with a nanodrop. These purified amplicons were used tooptimize real-time PCR conditions and to generate standard curves in thereal-time PCR assay. Primers were designed using Oligo 6 software.Primer concentrations and annealing temperatures were optimized for theICYCLER IQ™ system (Bio-Rad) for each set of primers using the system'sgradient protocol. PCR efficiencies were maintained between 92 and 105%and correlation coefficients >0.98 for each primer set duringoptimization and also during the real-time PCR of sample DNA. cDNAconcentrations for genes of interest were examined by real-time qPCRusing an ICYCLER IQ™ System and the IQ™ SYBR® Green Supermix (Bio-Rad).A standard curve was generated for each gene using 10-fold dilutions ofpurified amplicons starting at 5 pg of cDNA and used later to calculatethe starting amount of target cDNA in the unknown samples. SYBR® green Iis a general double-stranded DNA intercalating dye and may thereforedetect nonspecific products and primer/dimers in addition to theamplicon of interest. To determine the number of products synthesizedduring the real-time PCR, a melting curve analysis was performed on eachproduct. Real-time PCR was used to measure the starting amount ofnucleic acid of each unknown sample of cDNA on the same 96-well plate.

Statistical Analysis.

Parametric data were analyzed using the ANOVA followed by Scheffe'smultiple comparison method. Nonparametric data were analyzed by usingthe Mann-Whitney's U test followed by a Dunn's multiple comparisonstest. ANOVA was performed by using the general linear model procedure ofSAS, release 6.0.3 (SAS Institute). Statistical significance wasassessed at a P<0.05.

Results

BT-11 Improves Disease Activity in a Chronic IL-10−/− Model of IBD.

A number of animal studies to study the chronicity of Crohn's diseasehave employed the interleukin-10 deficient mice (IL-10−/−) mouse model,given that IL-10 is known to suppresses the secretion of numerousproinflammatory cytokines [21]. To assess the efficacy of BT-11 not onlyin acute models of colitis but also in chronic models, we set up anIL-10 null mouse model of colitis study and treated the mice withincreasing doses of BT-11 (20, 40, and 80 mg/Kg). Treatment with BT-11significantly decreased the disease activity index scores in treatedmice in comparison to their vehicle-treated littermates (FIG. 18).Furthermore, mice treated with the highest dose of BT-11 (80 mg/Kg)significantly reduced the scores in comparison to those treated witheither 20 or 40 mg/Kg of BT-11 compound starting at week 13 and untilthe end of the experiment.

BT-11 Reduced Macroscopic Lesions in Spleen, MLN, and Colon in anIL10−/− Chronic model of IBD.

To initially determine clinical efficacy we assessed macroscopic tissuelesion after treatment with BT-11 and subsequent LANCL2 pathwayactivation. We macroscopically scored the spleen (FIG. 19, panel A), theMLNs (FIG. 19, panel B), and the colon (FIG. 19, panel C) right aftereuthanasia and tissue collection 19 weeks after the start of the study.Treatment with BT-11 at concentrations as low as 20 mg/Kg greatly andsignificantly reduced the macroscopic scores in the three tissuesdemonstrating its potent efficacy.

BT-11 Improves Histopathological Lesions and Inflammation in a IL-O−/−Chronic Model of IBD.

To assess histopathological lesions and general pathology in the gutmucosa, colon sections were stained with H&E and observed under amicroscope. Our results show how treatment with BT-11 significantlyreduced inflammation based on the reduction of leukocytic infiltration(FIG. 20, panel A), epithelial erosion (FIG. 20, panel B), and mucosalthickening (FIG. 20, panel C). We also observed a dose-dependentmechanism on the amount of infiltration in the gut mucosa thatcorrelated to the thickening of the mucosa.

Treatment with BT-11 Induces a Potent Anti-Inflammatory Response andDecreases Pro-Inflammatory Subsets in the Colonic Lamina Propria,Spleen, and MLN.

To determine the effect of BT-11 on immune cell subsets, wephenotypically characterized cells isolated from the colon, spleen, andMLN. Our analyses indicated that BT-11 significantly decreased thepercentage of pro-inflammatory F4/80+ macrophages (FIG. 21, panel A),MHC-II+CD11c+ dendritic cells (FIG. 21, panel B), and effector Th1 cells(FIG. 21, panel D) in the colonic lamina propria. Furthermore, BT-11exerted anti-inflammatory properties via the upregulation ofFOXP3-expressing CD4+ T cells in the colonic LP (FIG. 21, panel C).

The upregulation of FOXP3-expressing CD4+ T cells was also noted in boththe MLN (FIG. 22, panel B) and the spleen (FIG. 22, panel C), showingand demonstrating how BT-11 has a systemic effect as well. Thedownregulation of pro-inflammatory Th1 cells was also observed in thespleen in a dose response manner (FIG. 22, panel D). Last, effector Th17cells, characterized by its expression of RORγt, were downregulated inthe MLN (FIG. 22, panel A).

Furthermore, gene expression analyses confirmed that treatment withBT-11 upregulates colonic expression of LANCL2 (FIG. 23, panel A) anddownregulates the expression of TNFα (FIG. 23, panel B). Theseexpression effects were dose-dependent on the amount of BT-11administered.

BT-11 Demonstrated Improvement of Disease Activity in a CD4+ T CellInduction of Colitis Model of IBD.

To further validate the efficacy of BT-11 in another chronic model ofIBD, we adoptively transferred naïve CD4+ T cells from wild-type andLANCL2−/− mice into RAG2−/− recipients. RAG2−/− mice were treated witheither vehicle or BT-11 based on experimental design. Treatment with ourtop lead compounds BT-11 significantly reduced the disease activityindex score in treated mice when compared to their wild-type littermates(FIG. 24). We found these results to be LANCL2-dependent as the effectof BT-11 was completely abrogated with the loss of LANCL2 (FIG. 25).

Interestingly, the weight loss in BT-11 treated mice was significantlyimproved when compared to vehicle treated mice starting at 7 weeks untilthe end of the experiment (FIG. 26).

BT-11 Reduced Macroscopic Lesions in Spleen, MLN, and Colon in anAdoptive Transfer Chronic Model of IBD.

To confirm the clinical efficacy in the second model of chronic colitiswe assessed macroscopic tissue lesion after treatment with BT-11 andsubsequent LANCL2 pathway activation in mice adoptively transferred withwild-type or LANCL2−/− cells and treated with either vehicle or BT-11.We macroscopically scored the spleen (FIG. 27, panel A), the MLNs (FIG.27, panel B), and the colon (FIG. 27, panel C) and the ileum (FIG. 27,panel D) right after euthanasia and tissue collection 11 weeks after thestart of the study. Treatment with BT-11 at a concentration of 80 mg/Kggreatly and significantly reduced the macroscopic scores in the fourtissues demonstrating its potent efficacy. We found these observationsto be LANCL2-dependent as well as the loss of LANCL2 completelyabrogated the effect of BT-11 (FIG. 28).

BT-11 Also Improves Histopathological Lesions and Inflammation in anAdoptive Transfer Model of Chronic Colitis.

Similar to the IL-10−/− induced colitis experiment and to confirmhistopathological lesions and general pathology in the gut mucosa with asecond mouse model of IBD, colon sections were stained with H&E andobserved under a microscope. Our results confirm how treatment withBT-11 significantly reduced inflammation based on the reduction ofleukocytic infiltration in both the colon and ileum (FIG. 29, panels Aand B) and mucosal thickening (FIG. 29, panels E and F). Of note, theileum was less affected on epithelial erosion (FIG. 29, panel D) butthat erosion in the colon was found significantly lower in mice treatedwith our top lead compound BT-11 (FIG. 29, panel C). To confirm thedependency to LANCL2 of BT-11, we performed adoptive transfer studiesand transferred CD4+ T cells from LANCL2−/− donors. Our results show howthe decrease in leukocytic infiltration, epithelial erosion, and mucosalthickening are greatly abrogated in LANCL2−/− transferred recipients(FIG. 30).

BT-11 Consistently Induces a Tremendous Anti-Inflammatory Response andDown-Regulates Pro-Inflammatory Mediators in Mice.

To characterize the immune cell profile in mice treated with BT-11versus vehicle, we performed flow cytometry analyses in cells isolatedfrom the colon, the spleen, and the mesenteric lymph nodes. We confirmedin a second chronic mouse model of IBD that recipient mice that weretreated with BT-11 for a period of 11 weeks possess a significantlylower level of infiltrating F4/80+CD11b+ pro-inflammatory macrophages(FIG. 31, panel A), as well as a decrease in IFNγ levels based on ananalysis made on total CD45+ leukocytes (FIG. 31, panel B) in the colon.Furthermore, treatment with BT-11 consistently upregulated regulatoryCD4+ T cells by promoting the expression of FOXP3 (FIG. 31, panel C) andthe potent anti-inflammatory cytokine IL-10 (FIG. 31, panel D) at thelocal site of inflammation, in this case, the colonic mucosa.

Similar to the profile observed in the colonic lamina propria cells, wecharacterized these populations in inductive sites such as the spleenand the MLN. Immuno-phenotyping results show how treatment with BT-11also increases the levels of FOXP3 and IL-10 in the inductive sites suchas spleen and MLN (FIG. 32, panels A, B, D, and E). Of note, thetreatment of BT-11 decreased the expression of IFNγ in the CD45+population in both the MLN and the spleen (FIG. 32, panels C and F).

The Effect of the LANCL2-Targeting BT-11 is Independent of PPARγ.

The activation of LANCL2 activates a plethora of pathways thatultimately regulate IL-10-based anti-inflammatory responses thatregulate inflammation at the systems level, based on our experimentalresults. One activated downstream pathway of LANCL2 is the PPARγpathway. To help overcome the potential toxicology concerns on thesecondary activation of this nuclear and transcription factor, we alsotransferred RAG2−/− mice with CD4+ T cells from PPARγ −/− donors. Wethen treated these mice with either vehicle or BT-11 at 80 mg/Kg. Ourresults clearly demonstrate that the beneficial effects of BT-11 viaactivation of LANCL2 on disease activity and histopathology occur in aPPARγ independent manner (FIG. 33, panels A-D). These resultsdemonstrate that the activation of LANCL2 also regulate other pathwaysthat modulate the anti-inflammatory effects of LANCL2 activation.

Discussion

Current therapies against inflammatory bowel disease (IBD) are modestlysuccessful and have significant adverse side effects for the long-termmanagement of the disease [17]. The botanical compound abscisic acid(ABA) exerts potent anti-inflammatory effects in mouse models of colitis[22, 23]. Lanthionine synthetase component C-like protein 2 (LANCL2) isa target for the binding and signaling of ABA [15, 19, 24]. Thus, LANCL2has emerged as a promising novel therapeutic target against inflammation[18]. Compound 61610, a bis(benzimidazoyl)terephthalanilide (BTT), wasidentified as binding to LANCL2 with the highest affinity in a libraryof several million chemicals. In addition, 61610 exerted potentanti-inflammatory effects in mouse models of gut inflammation [25]. Athematic library of 20 61610-derived BTTs were created and BT-11 wasidentified as a top exemplary compound. BT-11 binds to LANCL2, is orallyactive, has demonstrated anti-inflammatory efficacy in 3 mouse models ofcolitis and an outstanding safety profile.

According to the Crohn's and Colitis Foundation of America, IBD afflictsover 1 million people in North America and 4 million worldwide. Thiswidespread and debilitating illness results in decreased quality of lifeand significant health care-related costs [26]. Average medical expensesfor treating a single episode of IBD exceed $55,000 per patient [27]with total expenses exceeding $15 billion annually in the U.S. Inaddition, indirect expenses include the costs of treating recurrentpancreatitis [28] or other IBD complications such as abscesses,intestinal obstruction, anemia, thromboses, perianal lesions, arthritis,uveitis, iritis, or cutaneous lesions [29]. IBD carries a significantburden to patients, often isolating them socially, affecting familyrelationships and limiting their professional opportunities [17]. Inthis regard, patients with IBD have a higher rate of nonparticipation inthe labor force; this high rate persists over time [30]. In addition,intestinal inflammation (ulcerative colitis (UC) and Crohn's disease(CD)) increases the risk for developing colon cancer especially at earlyages (<30 years of age) [31]. The Global IBD Therapeutics Market isexpected to reach $4.3 Billion by 2015, according to a new report byGlobal Industry Analysts.

Even though current treatments for IBD have improved [17, 32], they areonly modestly successful for chronically managing the disease and resultin significant side effects, including a diminished ability of theimmune system to mount protective immune responses against pathogens ormalignancies. The treatment options for patients include addressing thesymptoms of inflammation. The majority of the pharmacological treatmentsused on the market today include aminosalicyclates, corticosteroids,immunomodulators, antibiotics, biologics (anti-tumor necrosisfactor-alpha antibody). Aminosalicyclates are extremely effective andgenerally well tolerated. However, patients with recurrences or moremoderate diseases may need more aggressive treatment, which includesshort-term doses of corticosteriods for a short period to control thesymptoms. This type of fast-acting therapy cannot be tolerated for longperiods. For maintenance of the condition, immunomodulators are alsocommonly used in CD and UC, but they have a slow onset of action (3 to 6months for the full effect). These medications have potentiallysignificant adverse side-effects ranging from pancreatitis, to diabetes,to scarred liver and inflamed lungs. For moderate to severe cases of thedisease that have failed management with other therapies, patients willbe placed on anti-TNF-α, which is given intravenously in a controlledsetting every 6-8 weeks. This extremely costly therapy, althougheffective, is difficult to access, as skilled personnel and a clinicalsetting are needed for administration. Further, significant side effectsexist such as Cushing's syndrome, mania, insomnia, hypertension, highblood glucose, osteoporosis, malignancies, infections, and avascularnecrosis of long bones.

The exemplary compound, BT-11, has shown a tremendously safe toxicologyprofile. Our efficacy data in chronic models of IBD show how treatmentof BT-11 improves disease activity scores in two models of chronic IBD(FIGS. 18 and 24) as well as body weight loss (FIG. 26). Our datademonstrates how these effects are LANCL2 dependent (FIG. 25). Ourefficacy data also demonstrates how activation of the LANCL2 pathway byBT-11 promotes an anti-inflammatory response mainly characterized byIL-10-producing and FOXP3-expressing CD4+ T cells (FIGS. 21, 22, 31, and32), as well as a significant decrease in inflammatory macrophages,dendritic cells, and pro-inflammatory factors such as IFNγ (FIGS. 22,23, 31, and 32). Moreover, the gene expression analyses confirm thesecell-based findings by showing how treatment with BT-11 reduces TNFαlevels in the colon (FIG. 23). All these findings together areresponsible for the dramatic LANCL2-dependent improvement in the colonicmucosa in terms of leukocytic infiltration, epithelial erosion, andmucosal thickening in two models of chronic IBD (FIGS. 20, 29, and 30).We have also demonstrated that the effects of BT-11 following binding toLANCL2 are PPARγ independent (FIG. 33). These results confirm that theactivation of LANCL2 activates a plethora of downstream activators thatregulate inflammation via a PPARγ independent mechanism. Together, theseresults strongly support the fact that LANCL2 is a novel therapeutictarget for inflammatory diseases and BT-11 is useful as a new drug.

Example 22: Use of BT-11 to Treat Type 1 Diabetes (T1D) Introduction

Diabetes mellitus (DM) also known as simply diabetes, is a group ofmetabolic diseases in which there are high blood sugar levels over aprolonged period of time. The two types of diabetes are referred to astype 1 and type 2. Former names for these conditions wereinsulin-dependent and non-insulin-dependent diabetes, or juvenile onsetand adult onset diabetes. In T1D the body does not produce insulin. Inrelation to T2D, T1D is nowhere near as common as T2D. Indeed,approximately 10% of all diabetes cases are type 1. T1D afflicts 3million Americans. Each year, more than 15,000 children and 15,000adults are diagnosed with T1D in the U.S. The rate of T1D incidenceamong children under age 14 is estimated to increase by 3% annuallyworldwide. T1D patients require insulin injections to stay alive, butthey do not cure the disease or prevent its serious side effects.

Current anti-diabetic medications are effective in improving insulinsensitivity, but their chronic administration has significant sideeffects such as cardiovascular complications, hepatotoxicity, weightgain, fluid retention, and bladder tumors. The lanthionine synthetasecomponent C-like 2 (LANCL2) pathway exerts anti-diabetic actions with noside effects [18]. BT-11 binds to LANCL2, is orally active, hasdemonstrated anti-diabetic efficacy in mice and an outstanding safetyprofile.

Methods

Mice.

NOD mice were purchased from the Jackson Laboratory and housed underspecific pathogen-free conditions in ventilated racks. The mice weremaintained in animal facilities. All experimental protocols wereapproved by an institutional animal care and use committee and met orexceeded guidelines of the National Institutes of Health Office ofLaboratory Animal Welfare and Public Health Service policy.

Assessment of Body Weight and Glucose Tolerance.

All mice were determined to be normoglycemic (fasting blood glucoselevels lower than 250 mg/dl) and to have similar weights (20±1.5 g)prior to the start of the study. Mice were weighed on a weekly basis andexamined for clinical signs of disease by blinded observers. After astandard 12 h fast, glucose was measured using an ACCU-CHEK® glucometer(Indianapolis, Ind.). Blood was collected via the lateral tail vein andplaced onto capillary blood collection tubes.

Histopathology.

Pancreatic sections from NOD studies in mice were fixed in 10% bufferedneutral formalin, later embedded in paraffin and then sectioned (5 am)and stained with H&E stain for histological examination. The sectionswere graded with a score of 0-4, depending on lymphocytic infiltration,cell damage and tissue erosion, and data were analyzed as a normalizedcompounded score.

Statistical Analysis.

Parametric data were analyzed using the ANOVA followed by Scheffe'smultiple comparison method. Nonparametric data were analyzed by usingthe Mann-Whitney's U test followed by a Dunn's multiple comparisonstest. ANOVA was performed by using the general linear model procedure ofSAS, release 6.0.3 (SAS Institute). Statistical significance wasassessed at a P≤0.05.

Results

BT-11 Lowers Fasting Blood Glucose Levels and Increases Insulin in aMouse Model of Type 1 Diabetes.

In order to determine the effect of BT-11 in modulating glycemic levelsin a mouse model of T1D, we performed a fasting blood glucose test onweeks 0, 1, 3, 4, 5, 10, and 11 after the start of the study. Ourresults show how the mice treated with our compound BT-11 hadsignificantly lower levels of glucose in blood after a period of 12 h offasting (FIG. 34, panel A). In parallel, we assessed insulin levels atweek 5 and our results show how mice treated with BT-11 hadsignificantly increased levels of insulin in plasma (FIG. 34, panel B).

BT-11 Improves Clinical Histopathological Pancreatic Lesions andInflammation in the Mouse NOD Model.

To assess histopathological lesions in the mouse model of T1D, pancreaswere collected and fixed with 10% formalin. Pancreatic sections werethen stained with H&E and observed under a microscope. Our results showhow treatment with BT-11 significantly reduces the clinicalhistopathological lesions in the pancreas in mice when compared to thevehicle treated mice (FIG. 35).

Discussion

There is a need for efficacious and safer oral medications for Type 1Diabetes (T1D), a disease that afflicts over 3 million Americans. ABAtreatment exerts anti-diabetic effects [2]. Lanthionine synthetasecomponent C-like protein 2 (LANCL2) is a target for the binding andsignaling of ABA [15, 19, 24]. Thus, LANCL2 has emerged as a promisingnovel therapeutic target against inflammation [18]. ABA is efficaciousin improving diabetes [2, 33] and immune-mediated diseases such asinflammatory bowel disease (IBD) [22, 23]. Compound 61610, abis(benzimidazoyl)terephthalanilide (BTT), binds to LANCL2 with thehighest affinity in a library of several million chemicals. In addition,61610 exerted potent immune modulatory effects in mouse models of gutinflammation [25]. BT-11 exerts anti-diabetic effects in NOD mice (FIGS.34 and 35). Moreover, ABA increased insulin secretion in humanpancreatic beta-cells [34], suggesting ABA's potential application asthe treatment of type 1 diabetes (T1D).

In immune cells, ABA is recognized by LANCL2, a G-protein couplereceptor that associates with the cell membrane following myristoylation[19, 35]. ABA binding to LANCL2 increases cAMP and initiates signalingthrough PKA and modulates immune responses in macrophages and T cells[8]. We performed homology modeling to construct a three-dimensionalstructure of LANCL2 by using the crystal structure of LANCL1 as atemplate. Using molecular docking, it was demonstrated first in silicoand then in vitro that ABA binds to LANCL2. This computationalprediction was validated by SPR results and a binding assay with humanLANCL2 [35]. We performed LANCL2-based virtual screening using thestructure of LANCL2 obtained through homology modeling to discover newLANCL2 ligands. Compounds from NCI Diversity Set II, ChemBridge and ZINCnatural products databases were docked into the LANCL2 model with AutoDock and ranked by the calculated affinity. While ABA has high affinityfor LANCL2, other diene-containing natural compounds such as 61610 werealso predicted to bind in the same region and can also be pursued asLANCL2-binding drugs [12]. BT-11 also has demonstrated strong binding toLANCL2 and therapeutic efficacy in the NOD mouse model of T1D (FIG. 34).This data provides some validation that the LANCL2 pathway and the othercompounds of the invention are useful as immune modulatory drugs forT1D. Further evidence in support of the role of the LANCL2 pathway as ameans of modulating immune responses and ameliorating autoimmunediseases includes the LANCL2 binding and protective effects of ABA [22,23], 61610 [12, 18] and BT-11 in mouse models of inflammatory boweldisease (IBD).

The incidence of T1D is increasing at an estimated annual rate of 3%worldwide [36-38]. While successful transplantation of pancreatic isletscan treat T1D, the lack of sufficient islets, ongoing immune-mediateddestruction of transplanted islets, and side effects from theimmunosuppressive drugs greatly limits the widespread use of thisapproach [39]. As such, therapies that safely combine the ability ofpromoting pancreatic β-cell function and immune modulation arefundamental strategies to treat T1D. Our data demonstrate thatactivation of LANCL2 by BT-11 not only improves glucose levels in blood,but also improves its normalization after a glucose challenge (FIG. 34).Furthermore, treatment with BT-11 during the onset of T1d improveshistopathology in the pancreas (FIG. 35). Indeed, ABA preventively andtherapeutically suppresses inflammation and improves glucose tolerance[2, 3]. Thus, the natural activation of LANCL2 results in both immunemodulation as illustrated by its therapeutic effects in IBD [12, 18, 22,23] and regulation of glucose homeostasis due to suppressed inflammationand enhanced insulin sensitivity [2, 3]. Based on this background anddata presented in FIGS. 34 and 35, investigating the role of LANCL2 as atherapeutic target for T1D is important.

Example 23: Use of BT-11 to Treat Type 2 Diabetes (T2D) Introduction

Diabetes mellitus (DM) is a chronic condition that occurs when the bodycannot produce enough or effectively use of insulin, and are induced bya genetic predisposition coupled with environmental factors. Unlikepeople with type 1 diabetes, type 2 diabetics are able to produceinsulin. However, the pancreas of such patients does not make enoughinsulin or the body cannot use the insulin well enough. This phenomenais called insulin resistance. When there isn't enough insulin or theinsulin is not used as it should be, glucose cannot be processed andused. As a result, when glucose is accumulated in the blood streaminstead of going into cells and being metabolized, other cells in thesystem cannot function properly. Indeed, hyperglycemia and diabetes areimportant causes of morbidity and mortality, due to cardiovasculardisease (CVD), nephropathy, neuropathy, foot ulcers, and retinopathy.

About 28.3 million Americans have type 2 diabetes (T2D) and over 40.1%of middle-aged adults have pre-diabetes, a condition characterized byimpaired glucose tolerance, systemic inflammation and insulinresistance. The World Health Organization estimates that the number ofpeople with T2D will increase to 366 million by the year 2030.

As stated above, current anti-diabetic medications are effective inimproving insulin sensitivity, but their chronic administration hassignificant side effects such as cardiovascular complications,hepatotoxicity, weight gain, fluid retention, and bladder tumors. Thelanthionine synthetase component C-like 2 (LANCL2) pathway exertsanti-diabetic actions with no side effects [18]. BT-11 binds to LANCL2,is orally active, has demonstrated anti-diabetic efficacy in mice and anoutstanding safety profile.

Methods

Mice and Dietary Treatments.

C57BL/6 and db/db, mice were purchased from the Jackson Laboratory andhoused under specific pathogen-free conditions in ventilated racks. Micein the Diet Induced Obesity diabetes model (DIO) were fed a high-fatdiet (40 Kcal % fat). The mice were maintained in animal facilities. Allexperimental protocols were approved by an institutional animal care anduse committee and met or exceeded guidelines of the National Institutesof Health Office of Laboratory Animal Welfare and Public Health Servicepolicy.

Assessment of Body Weight and Glucose Tolerance.

All mice were determined to be normoglycemic (fasting blood glucoselevels lower than 250 mg/dl) and to have similar weights (weight±1.5 g)prior to the start of the study. Mice were weighed on a weekly basis andexamined for clinical signs of disease by blinded observers. After astandard 12 h fast, glucose was determined on different days. Briefly,blood was collected via the lateral tail vein and placed onto capillaryblood collection tubes. Mice then were administered a glucose tolerancetest by intraperitoneal injection of D-glucose (2 g/kg body weight) andblood samples collected prior to the injection (time 0) (correspondingto a baseline FBG level following a 12-h fast starting at 6 a.m.) and at15, 60, and 90 minutes (db/db model) or 15, 30, 60, 90, 120, 180, 220,and 265 minutes (DIO model) following the glucose injection. Abdominal(epididymal) white adipose tissue (WAT), subcutaneous WAT, and liverwere then excised and weighed. Abdominal (epididymal) WAT was thendigested and fractionated.

Digestion of White Adipose Tissue.

Abdominal WAT was excised, weighed, minced into small <10 mg pieces andplaced into digestion media (1×HBSS (Mediatech, Herndon, Va.)supplemented with 2.5% HEPES (Mediatech) and 10% fetal bovine serumcontaining type II collagenase (0.2%, Sigma-Aldrich)). Samples wereincubated in a 37° C. incubator for 30 min, filtered through a 100 mnylon cell strainer to remove undigested particles, and centrifuged at4° C. at 1000×g for 10 min. The pellet, consisting of stromal vascularcells (SVCs), was washed with 1×HBSS and centrifuged at 4° C. at 1000×gfor 10 min. The supernatant was discarded and erythrocytes were lysed byincubating the SVCs in 2 mL erythrocyte lysis buffer for 2 min beforestopping the reaction with 9 mL 1×PBS. Cells were then respun at 4° C.at 1000×g for 10 min, suspended in 1 mL of 1×PBS, and counted with aCoulter Counter (Beckman Coulter, Fullerton, Calif.).

Immunophenotyping of Stromal Vascular Cells.

For immunophenotyping SVCs were seeded into 96-well plates (Costar) at2×105 cell/well. After an initial 20 min incubation with FcBlock (20μg/mL; BD Biosciences—Pharmingen) to inhibit non-specific binding, cellswere washed in PBS containing 5% serum and 0.09% sodium azide (FACSbuffer) and stained with specific primary anti-mouse antibodies. Flowresults were computed with a FacsAria flow cytometer and data analyseswere performed with FACS DIVA™ (BD Biosciences) and FlowJo (TreeStar).

RealTime Quantitative PCR.

Total RNA was isolated from adipose tissue using the RNEASY Lipid MiniKit (Qiagen) and from cells using the RNEASY Mini Kit (Qiagen) accordingto the manufacturer's instructions. Total RNA was used to generatecomplementary DNA (cDNA) template using the QSCRIPT™ cDNA Synthesis Kit(Quanta Biosciences, Gaithersburg, Md.). The total reaction volume was20 μL with the reaction incubated as follows in an MJ MINI™ thermalcycler (Bio-Rad): 5 min at 25° C., 30 min at 52°, 5 min at 85° C., holdat 4° C. Each gene amplicon was purified with the MINELUTE PCRPurification Kit (Qiagen) and quantitated on an agarose gel by using aDNA mass ladder (Promega). These purified amplicons were used tooptimize real-time PCR conditions in the real-time PCR assay. Primerconcentrations and annealing temperatures were optimized for the CFXsystem (Bio-Rad) for each set of primers using the system's gradientprotocol. PCR efficiencies were maintained between 92 and 105% andcorrelation coefficients above 0.98 for each primer set duringoptimization and also during the real-time PCR of sample DNA. Data isshown using the ΔΔCt quantification method.

Results

BT-11 Reduced Fasting Blood Glucose Levels in a Mouse DIO Model of T2D.

To assess the efficacy of the exemplary compound BT-11 in a model ofT2D, we fed C57BL/6 mice a high fat diet (DIO model). Oral BT-11administration significantly decreased the levels of blood glucose inBT-11 treated mice when compared to their vehicle-treated littermates atweek 12 of high-fat feeding (FIG. 36, panel A). Furthermore, after 12 hfasting and glucose challenge at 2 g/Kg body weight via IP, mice treatedwith BT-11 were capable to normalize blood glucose levels significantlyfaster than untreated mice (FIG. 36, panel B).

BT-11 Treatment Decreased Pro-Inflammatory Macrophage Infiltration asWell as Pro-Inflammatory Granulocytes in White Adipose Tissue.

In order to characterize the cells infiltrating the white adiposetissue, abdominal WAT was collected and digested as specified in themethods section. Flow cytometry analyses were performed evaluatingdifferent pro-inflammatory populations in WAT. Our results show howtreatment with BT-11 significantly reduced the levels of F4/80+CD11b+pro-inflammatory macrophages (FIG. 37, panel A), as well as the numberof pro-inflammatory granulocytes with high levels of Ly6c (GR1+Ly6chigh)(FIG. 37, panel B).

BT-11 Reduced Fasting Blood Glucose Levels in a Mouse Db/Db Model ofT2D.

To evaluate the therapeutic efficacy of oral BT-11 treatment in twomouse models of diabetes, we also used the db/db mice, which developsspontaneous T2D due to a mutation in the leptin receptor. Db/db micewere administered a daily dose of BT-11 at 80 mg/Kg by oral gavage. Wedetermined the effect of BT-11 on glucose homeostasis by measuringfasting blood glucose concentrations. Treatment with BT-11 significantlydecreased the levels of blood glucose in comparison to theirvehicle-treated littermates as early as in one week, accentuating thedifferences over time at week 3 (FIG. 38, panel A). To determine whetheroral BT-11 treatment modulates how the animal initiates glucosehomeostasis, we gave an intraperitoneal glucose challenge toexperimental animals and evaluated the kinetics of plasma glucose from 0to 265 minutes following glucose injection. Blood samples collectedprior to the injection (time 0) (corresponding to a baseline FBG levelfollowing a 12-h fast). Our results show how oral treatment with BT-11significantly decreases the levels of glucose prior to the IP glucosechallenge (Time 0, FIG. 38, panel B). Following glucose challenge in thedb/db model, our results show how glucose levels in mice treated withour top lead compounds BT-11, fell toward normal levels more rapidlythan in the vehicle-treated mice (FIG. 38, panel B).

BT-11 Reduced mRNA Levels of TNFα and MCP-1 and Upregulated LANCL2.

To further confirm the anti-inflammatory potency of BT-11, we assessedgene expression on WAT as indicated in the methods section. Our resultsshow how when compared to untreated mice, mice treated with BT-11 havehigher expression levels of LANCL2 and significantly lower mRNA levelsof the pro-inflammatory factor TNFα and MCP-1 (FIG. 39).

Discussion

As the rates of obesity and Type 2 Diabetes (T2D) in the U.S. continueto rise, an increasingly large number of people are becoming reliant onoral anti-diabetic drugs. About 28.3 million (8.3% of the population)Americans have T2D and over 40.1% of middle-aged adults hadpre-diabetes, a condition characterized by impaired glucose toleranceand insulin resistance [40]. The total direct and indirect costsattributable to T2D in the United States are over $132 billion [40].Despite this growing problem, pharmaceutical manufacturers have beenunable to develop medications that are both safe and effective. One ofthe most popular and effective oral anti-diabetic medications is thethiazolidinedione (TZD) class of insulin-sensitizing drugs. AlthoughTZDs enhance insulin sensitivity, they have significant adverse sideeffects that have limited their availability, including weight gain,congestive heart failure, bladder cancer, hepatotoxicity and fluidretention [41, 42]. For instance, approximately 10-15% of patients usingTZDs are forced to discontinue treatment due to edema, and the increasein extracellular volume from excess fluid retention also poses a majorproblem for individuals with preexisting congestive heart failure. In2000, troglitazone (REZULIN®) was removed from the market, 3 years afterits inception, due to reports of serious liver injury and death [43].Safety concerns about other TZDs resulted in mandatory black boxlabeling and subsequent restrictions for use.

LANCL2 was the second member of the LanC-like protein family to beidentified. The first member, LANCL1, was isolated from humanerythrocyte membranes [44]. LANCL2 was subsequently identified andexpressed throughout the body [1, 18], including immune cells, pancreas,lung and intestine [1, 44]. The lanthionine synthetase C-like 2 (LANCL2)pathway has emerged as a novel therapeutic target for T2D [18].Extensive pre-clinical testing provides ample evidence of thetherapeutic potential for LANCL2 ligands such as abscisic acid (ABA) indiabetes and chronic inflammatory diseases [2, 3, 22, 23, 45]. Compound61610, a bis(benzimidazoyl)terephthalanilide (BTT) binds to LANCL2 withthe highest affinity in a library of several million chemicals.

Given the fact that current drugs for T2D fail to satisfy the patientfirst need, which is glycemic control, without side effects, BT-11represents a very attractive potential substitute. Our results show howthe administration of BT-11 in different mouse models of T2Dsignificantly lowers the glucose levels in blood after a period offasting (FIGS. 36 and 38). Moreover, the administration of this compoundalso helps normalize glucose levels after a glucose challenge (FIGS. 36and 38). The anti-inflammatory properties of BT-11 are also reflected inour immunophenotyping results. Indeed, administration of BT-11 resultedin less infiltration of pro-inflammatory macrophages andpro-inflammatory granulocytes in abdominal WAT (FIG. 37). These resultswere supported by gene expression data of two very importantpro-inflammatory factors, TNFα and MCP-1, which were found significantlyreduced in mice treated with BT-11 (FIG. 39).

Example 24: Use of BT-11 During Influenza Infection Introduction

Respiratory pathogens causing pneumonia are the leading cause ofinfectious disease-related death in industrialized countries. Theabsence of effective vaccines and anti-virals coupled with growingconcerns over the emergence of anti-viral resistance highlights a needfor developing host-targeted immunotherapeutic approaches. The pulmonarypathogenesis and clinical disease associated with respiratory infectionsoften result from a combination of the cytopathic effects of the virusand the host immune response. In this regard, therapies directed atmodulating the innate immune response are considered for the treatmentof flu [46].

Influenza remains a major public health problem worldwide. Seasonalinfluenza is associated with an upper respiratory tract process which isoften incapacitating and requires days of restricted activity. It hasbeen estimated that in the United States alone, annual flu epidemicsresult in 30 million outpatient visits and 300,000 hospital admissions.Certain populations (e.g., young children, the elderly, and people withpredisposing medical conditions) are at higher risk of developing viralpneumonia. Experts have estimated that 25,000 to 35,000 people dieannually from seasonal flu in the US, and the global financial burdenhas been calculated to be hundreds of billions of dollars [47]. Pandemicinfluenza cycles occur every 30-50 years with added complexity due totheir unpredictable presentation and lack of pre-existing immunity, andare associated with high mortality rates [48]. Influenza is associatedwith significant morbidity and mortality, but effective and safe drugtreatments are lacking.

Data that suggests lanthionine synthetase component C-like protein 2(LANCL2) is a target for the binding and signaling of ABA [15, 19, 24].Thus, LANCL2 has emerged as a promising novel therapeutic target forimmune modulation. Using molecular modeling and surface plasmonresonance (SPR), BTI has identified compound BT-11, abis(benzimidazoyl)terephthalanilide (BTT), which binds to LANCL2 withhigh affinity. Also, BT-11 exerted potent pro-resolutive effects in thelungs, and decreased mortality and morbidity in mouse models ofinfluenza.

Methods

Mice.

C57BL/6 mice were purchased from the Jackson Laboratory and housed underspecific pathogen-free conditions in ventilated racks. All experimentalprotocols were approved by an institutional animal care and usecommittee and met or exceeded guidelines of the National Institutes ofHealth Office of Laboratory Animal Welfare and Public Health Servicepolicy.

Intranasal Infection of Mice with Influenza Virus.

Mice were anesthetized with 2-5% isofluorane using a vaporizer station,and 50 μL of virus dilution at 10³ TCID50 was administered through thenostrils (25 μL each one). Mice were then placed in their cages andwatched for recovery of anesthesia.

Oral Administration of BT-11 by Orogastric Gavage.

BT-11 was administered to mice by orogastric gavage using a commerciallyavailable safety ball-tipped gavage needle (18-24 gauge, depending onthe weight of the animal). This procedure caused no pain or distress.Mice were treated with BT-11 at a dose of 80 mg/Kg every 24 h for theduration of the experiment.

Monitoring of Mice and Disease Activity and Weighing.

Mice were monitored once daily after the infection (or every 4 hours ifthey developed severe clinical signs of disease equivalent to diseasescore 2) and were euthanized prior to the planned endpoint if theydeveloped significant signs of illness as measured by weight loss (i.e.,25% gradual loss of initial body weight), dehydration, loss of mobility,guarding/protection of painful area, ruffled fur (piloerection). Micewere weighed once a day for the duration of the experiment.

Results Oral Administration of BT-11 Reduced Clinical Scores andMorbidity in Mice with Influenza Virus.

To evaluate the therapeutic efficacy of BT-11, we used a mouse model ofinfluenza infection in mice. Briefly, mice were infected intranasallyafter anesthesia with 5% isofluorane. Mice were daily treated with anoral suspension of BT-11 at 80 or 40 mg/Kg. Mice were weighed and scoredfor the duration of the experiment (16 days). Results show howadministration of BT-11 significantly reduced the activity clinicalscore starting at day 3 and throughout the experiment (FIG. 40, panelA). Furthermore, the clinical score for physical appearance wassignificantly reduced in mice receiving the treatment with both 40 and80 mg/Kg of BT-11 (FIG. 40, panel B).

In order to evaluate the effect of the treatment in disease morbidity,we calculated the percentage of weight loss and further evaluated thenumber of mice losing over 15% within each experimental group. Startingat day 6 post-infection, the treatment with 80 mg/Kg of BT-11 resultedin less morbidity when comparing to the vehicle group. The differencesaccentuated starting at day 10 and through day 12 (FIG. 40, panel C).

Discussion

Traditional approaches to control influenza spread and disease arecentered on the virus side through vaccination and antiviral treatment.Vaccines have to be formulated annually based on the circulating strainsfrom the previous season. However it takes about 4 to 6 months toproduce, license, and test the efficacy of a new vaccine [49], whetherit is for seasonal or pandemic flu. The main disadvantage of antiviralsis the very frequent emergence and selection of resistant strains. Inaddition to virus-centered treatments, the development of therapiesbased on controlling exacerbated host responses have a very highlikelihood of being adopted to complement anti-microbial andprophylactic strategies. Host-targeted therapeutics have the advantageof offering cross protection among different reasortants, and thus beingefficacious from season to season, they can be produced and stocked, andcan be used to treat the disease after virus exposure [46, 50, 51].

The identification of LANCL2 as a novel therapeutic target for influenzaopens a new avenue for host-targeted therapeutics. We demonstrated thatactivation of LANCL2 by BT-11 improves not only activity and clinicalscores, but also decreases morbidities caused by the influenza virus,and accelerates the recovery from influenza infection (FIG. 33). Theseresults strongly support that LANCL2 is a novel therapeutic target forinfluenza and BT-11 is a potential new host-targeted drug.

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1-50. (canceled)
 51. A composition comprising: a compound of formula:

or a pharmaceutically acceptable salt thereof, wherein: Q ispiperazine-1,4-diyl or substituted piperazine-1,4-diyl wherein thepiperazine in the substituted piperazine-1,4-diyl is substituted withone to eight substituents in the 2, 3, 5, or 6 positions and wherein thesubstituents are independently selected from the group consisting of (C₁to C₆)alkyl, aryl, aryl(C₁ to C₆)alkyl, C(O)OH, and C(O)O(C₁ toC₆)alkyl; A₁ and A₁′ are each independently N or CR⁶; A₂ and A₂′ areeach independently N or CR⁷; A₃ is NR⁸; A₃′ is NR⁸, O, or S; A₄ and A₄′are each independently N or CR⁹; A₅ and A₅′ are each independently N orCR¹⁰; A₆ and A₆′ are each independently N or CR¹¹; and R¹, R^(1′), R²,R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹, ifpresent, are in each instance independently selected from the groupconsisting of hydrogen, alkyl, halo, trifluoromethyl, dialkylaminowherein each alkyl is the same or different, —NH₂, alkylamino, andarylalkyl; and a carrier.
 52. The composition of claim 51, wherein R¹,R^(1′), R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, andR¹¹, if present, are in each instance independently selected from thegroup consisting of hydrogen, alkyl, halo, and trifluoromethyl.
 53. Thecomposition of claim 51, wherein R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴,R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹, if present, are each hydrogen. 54.The composition of claim 51, wherein: A₃′ is NR⁸ or O; A₄ and A₄′ areeach N; A₅ and A₅′ are each independently CR¹⁰; and A₆ and A₆′ are eachindependently CR¹¹.
 55. The composition of claim 54, wherein R¹, R^(1′),R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹, ifpresent, are in each instance independently selected from the groupconsisting of hydrogen, alkyl, halo, and trifluoromethyl.
 56. Thecomposition of claim 54, wherein R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴,R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹, if present, are each hydrogen. 57.The composition of claim 51, wherein Q is piperazine-1,4-diyl.
 58. Thecomposition of claim 57, wherein R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴,R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹, if present, are in each instanceindependently selected from the group consisting of hydrogen, alkyl,halo, and trifluoromethyl.
 59. The composition of claim 57, wherein R¹,R^(1′), R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, andR¹¹, if present, are each hydrogen.
 60. The composition of claim 57,wherein: A₃′ is NR⁸ or O; A₄ and A₄′ are each N; A₅ and A₅′ are eachindependently CR¹⁰; and A₆ and A₆′ are each independently CR¹¹.
 61. Thecomposition of claim 60, wherein R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴,R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹, if present, are in each instanceindependently selected from the group consisting of hydrogen, alkyl,halo, and trifluoromethyl.
 62. The composition of claim 60, wherein R¹,R^(1′), R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, andR¹¹, if present, are each hydrogen.
 63. The composition of claim 60,wherein: A₁ and A₁′ are each N; and A₂ and A₂′ are each independentlyCR⁷.
 64. The composition of claim 63, wherein R¹, R^(1′), R², R^(2′),R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹, if present, are ineach instance independently selected from the group consisting ofhydrogen, alkyl, halo, and trifluoromethyl.
 65. The composition of claim63, wherein R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸,R⁹, R¹⁰, and R¹¹, if present, are each hydrogen.
 66. The composition ofclaim 51, wherein the compound has the structure of:

or a salt thereof.
 67. The composition of claim 51, wherein the compoundhas the structure of:

or a salt thereof.
 68. The composition of claim 51, wherein the compoundhas the structure of:

or a salt thereof.
 69. A method of treating inflammatory bowel diseasein an animal, the method comprising administering an effective amount ofa composition to the animal, wherein the composition comprises: acompound of formula:

or a pharmaceutically acceptable salt thereof, wherein: Q ispiperazine-1,4-diyl or substituted piperazine-1,4-diyl wherein thepiperazine in the substituted piperazine-1,4-diyl is substituted withone to eight substituents in the 2, 3, 5, or 6 positions and wherein thesubstituents are independently selected from the group consisting of (C₁to C₆)alkyl, aryl, aryl(C₁ to C₆)alkyl, C(O)OH, and C(O)O(C₁ toC₆)alkyl; A₁ and A₁′ are each independently N or CR⁶; A₂ and A₂′ areeach independently N or CR⁷; A₃ is NR⁸; A₃′ is NR⁸, O, or S; A₄ and A₄′are each independently N or CR⁹; A₅ and A₅′ are each independently N orCR¹⁰; A₆ and A₆′ are each independently N or CR¹¹; and R¹, R^(1′), R²,R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶, R⁶, R⁸, R⁹, R¹⁰, and R¹¹, ifpresent, are in each instance independently selected from the groupconsisting of hydrogen, alkyl, halo, trifluoromethyl, dialkylaminowherein each alkyl is the same or different, —NH₂, alkylamino, andarylalkyl; and a carrier.
 70. The method of claim 69, wherein R¹,R^(1′), R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, andR¹¹, if present, are in each instance independently selected from thegroup consisting of hydrogen, alkyl, halo, and trifluoromethyl.
 71. Themethod of claim 69, wherein R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴,R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹, if present, are each hydrogen. 72.The method of claim 69, wherein: A₃′ is NR⁸ or O; A₄ and A₄′ are each N;A₅ and A₅′ are each independently CR¹⁰; and A₆ and A₆′ are eachindependently CR¹.
 73. The method of claim 72, wherein R¹, R^(1′), R²,R₂′, R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹, if present,are in each instance independently selected from the group consisting ofhydrogen, alkyl, halo, and trifluoromethyl.
 74. The method of claim 72,wherein R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸, R⁹,R¹⁰, and R¹¹, if present, are each hydrogen.
 75. The method of claim 69,wherein Q is piperazine-1,4-diyl.
 76. The method of claim 75, whereinR¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, andR¹¹, if present, are in each instance independently selected from thegroup consisting of hydrogen, alkyl, halo, and trifluoromethyl.
 77. Themethod of claim 75, wherein R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴,R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹, if present, are each hydrogen. 78.The method of claim 75, wherein: A₃′ is NR⁸ or O; A₄ and A₄′ are each N;A₅ and A₅′ are each independently CR¹⁰; and A₆ and A₆′ are eachindependently CR¹¹.
 79. The method of claim 78, wherein R¹, R^(1′), R²,R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹, ifpresent, are in each instance independently selected from the groupconsisting of hydrogen, alkyl, halo, and trifluoromethyl.
 80. The methodof claim 78, wherein R¹, R^(1′), R², R², R³, R^(3′), R⁴, R^(4′), R⁶, R⁷,R⁸, R⁹, R¹⁰, and R¹¹, if present, are each hydrogen.
 81. The method ofclaim 78, wherein: A₁ and A₁′ are each N; and A₂ and A₂′ are eachindependently CR⁷.
 82. The method of claim 81, wherein R¹, R^(1′), R²,R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶, R⁷, R⁸, R⁹, R¹⁰, and R¹¹, ifpresent, are in each instance independently selected from the groupconsisting of hydrogen, alkyl, halo, and trifluoromethyl.
 83. The methodof claim 81, wherein R¹, R^(1′), R², R^(2′), R³, R^(3′), R⁴, R^(4′), R⁶,R⁷, R⁸, R⁹, R¹⁰, and R¹¹, if present, are each hydrogen.
 84. The methodof claim 69, wherein the compound has the structure of:

or a salt thereof.
 85. The method of claim 84, wherein the inflammatorybowel disease is ulcerative colitis.
 86. The method of claim 84, whereinthe inflammatory bowel disease is Crohn's disease.
 87. The method ofclaim 69, wherein the compound has the structure of:

or a salt thereof.
 88. The method of claim 69, wherein the compound hasthe structure of:

or a salt thereof.
 89. The method of claim 69, wherein the inflammatorybowel disease is ulcerative colitis.
 90. The method of claim 69, whereinthe inflammatory bowel disease is Crohn's disease.